Chapter 12 · Campbell Essential Biology, ... Chapter 12 DNA Technology. Biology and Society: DNA, Guilt, and Innocence ... Figure 12.8 Plasmid Bacterial cell 1 Isolate plasmids

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Chapter 12DNA Technology

20_02SeaLion_SV.mpg

Biology and Society: DNA, Guilt, and Innocence

• DNA profiling is the analysis of DNA samples that

can be used to determine whether the samples

come from the same individual.

• DNA profiling can therefore be used in courts to

indicate if someone is guilty of a crime.

© 2013 Pearson Education, Inc.

Figure 12.0

• DNA technology has led to other advances in the

– creation of genetically modified crops and

– identification and treatment of genetic diseases.

Biology and Society: DNA, Guilt, and Innocence


RECOMBINANT DNA TECHNOLOGY

• Biotechnology

– is the manipulation of organisms or their

components to make useful products and

– has been used for thousands of years to

– make bread using yeast and

– selectively breed livestock for desired traits.


• Biotechnology today means the use of DNA

technology, techniques for

– studying and manipulating genetic material,

– modifying specific genes, and

– moving genes between organisms.



• Recombinant DNA is constructed when scientists

combine pieces of DNA from two different sources

to form a single DNA molecule.

• Recombinant DNA technology is widely used in

genetic engineering, the direct manipulation of

genes for practical purposes.



Figure 12.1

Applications: From Humulin to Foods to “Pharm” Animals

• By transferring the gene for a desired protein into a

bacterium or yeast, proteins that are naturally

present in only small amounts can be produced in

large quantities.


Making Humulin

• In 1982, the world’s first genetically engineered

pharmaceutical product was sold.

• Humulin, human insulin

– was produced by genetically modified bacteria and

– is used today by more than 4 million people with

diabetes.

• Today, humulin is continuously produced in

gigantic fermentation vats filled with a liquid culture

of bacteria.


Figure 12.2

Figure 12.3

• DNA technology is used to produce medically

valuable molecules, including

– human growth hormone (HGH),

– the hormone erythropoietin (EPO), which

stimulates production of red blood cells, and

– vaccines, harmless variants or derivatives of a

pathogen used to prevent infectious diseases.

Making Humulin


Genetically Modified (GM) Foods

• Today, DNA technology is quickly replacing

traditional breeding programs.

• Scientists have produced many types of

genetically modified (GM) organisms, organisms

that have acquired one or more genes by artificial

means.

• A transgenic organism contains a gene from

another organism, typically of another species.


• In the United States today, roughly half of the corn

crop and more than three-quarters of the soybean

and cotton crops are genetically modified.

• Corn has been genetically modified to resist insect

infestation, attack by an insect called the European

corn borer.



Figure 12.4

• Strawberry plants produce bacterial proteins that

act as a natural antifreeze, protecting the plants

from cold weather.

• Potatoes and rice have been modified to produce

harmless proteins derived from the cholera

bacterium and may one day serve as edible

vaccines.



• ―Golden rice 2‖

– is a transgenic variety of rice that carries genes from daffodils and corn and

– could help prevent vitamin A deficiency and resulting blindness.



Figure 12.5

“Pharm” Animals

• A transgenic pig has been produced that carries a gene for human hemoglobin, which can be

– isolated and

– used in human blood transfusions.

• In 2006, genetically modified pigs carried roundworm genes that produce proteins that convert less healthy fatty acids to omega-3 fatty acids.

• However, unlike transgenic plants, no transgenic animals are yet sold as food.


Figure 12.6

Recombinant DNA Techniques

• Bacteria are the workhorses of modern

biotechnology.

• To work with genes in the laboratory, biologists

often use bacterial plasmids, small, circular DNA

molecules that replicate separately from the larger

bacterial chromosome.


Figure 12.7

Plasmids

Bacterial

chromosome

Remnant of

bacterium

Co

lori

ze

d T

EM

• Plasmids

– can carry virtually any gene,

– can act as vectors, DNA carriers that move genes

from one cell to another, and

– are ideal for gene cloning, the production of

multiple identical copies of a gene-carrying piece of

DNA.



• Recombinant DNA techniques can help biologists

produce large quantities of a desired protein.



Figure 12.UN01DNA isolated from two sources and cut by samerestriction enzyme

Gene of interest(could be obtained froma library or synthesized)

RecombinantDNA

Plasmid(vector)

Transgenic organisms

Useful products

Figure 12.8

Plasmid

Bacterial cellIsolate plasmids.1 2

3

4

5

6

7

Cut both DNAs

with same

enzyme.

Isolate DNA.

Gene

of

interest

Other

genes

DNA fragments

from cell

DNA

Cell containing

the gene of interest

Mix the DNA fragments and join them together.

Gene of interest

Recombinant DNA plasmids

Bacteria take up recombinant plasmids.

Recombinant bacteria

Bacterial cloneClone the bacteria.

Find the clone with the gene of interest.

A protein is used to

dissolve blood clots

in heart attack

therapy.

A protein is used to prepare

“stone-washed” blue jeans.

Bacteria

produce

proteins,

which can be harvested

and used directly.

The gene

and protein

of interest

are isolated

from the

bacteria.

Genes may

be inserted

into other

organisms.

Some uses

of genes

Some uses

of proteins

A gene for pest

resistance is

inserted into

plants.

A gene is used to alter

bacteria for cleaning

up toxic waste.

8

Figure 12.8c

8

Protein for

dissolving

clots

Protein for

“stone-washing”

jeans

Harvested

proteins

may be

used

directly.

The gene

and protein

of interest

are isolated

from the

bacteria.

Genes may

be inserted

into other

organisms.

Some uses

of genes

Some uses

of proteins

Gene

for pest

resistance

Genes for

cleaning up

toxic waste

A Closer Look: Cutting and Pasting DNA with Restriction Enzymes

• Recombinant DNA is produced by combining two

ingredients:

1. a bacterial plasmid and

2. the gene of interest.

• To combine these ingredients, a piece of DNA

must be spliced into a plasmid.


• This splicing process can be accomplished by

– using restriction enzymes, which cut DNA at

specific nucleotide sequences (restriction sites),

and

– producing pieces of DNA called restriction

fragments with ―sticky ends‖ important for joining

DNA from different sources.



• DNA ligase connects the DNA pieces into

continuous strands by forming bonds between

adjacent nucleotides.



Figure 12.9-4

Recognition site (recognition sequence)

for a restriction enzyme

Restriction

enzyme

DNA

DNA

ligase

Recombinant DNA molecule

A DNA fragment is added from

another source.

A restriction enzyme cuts the

DNA into fragments.

Fragments stick together by

base pairing.

DNA ligase joins the fragments

into strands.

1

2

3

4

A Closer Look: Obtaining the Gene of Interest

• How can a researcher obtain DNA that encodes a

particular gene of interest?

– A ―shotgun‖ approach can yield millions of

recombinant plasmids carrying many different

segments of foreign DNA.

– A collection of cloned DNA fragments that includes

an organism’s entire genome (a complete set of its

genes) is called a genomic library.


• Once a genomic library is created, the bacterial

clone containing the desired gene is identified

using a nucleic acid probe consisting of a short

single strand of DNA with a complementary

sequence and labeled with either a radioactive

isotope or a fluorescent dye.



Figure 12.10

Radioactive probe(single-stranded DNA)

Single-stranded DNA

Mix with single-stranded DNA from various bacterial clones

Base pairing indicates the gene of interest

• Another way to obtain a gene of interest is to

– use reverse transcriptase and

– synthesize the gene by using an mRNA template.



Figure 12.11

Cell nucleus

DNA ofeukaryoticgene

Test tube

Transcription

Exon Intron Exon ExonIntron

RNA

transcript

mRNA

Introns removed

and exons spliced

together

Isolation of mRNA

from cell and

addition of

reverse transcriptase

Synthesis of cDNA

strand

Synthesis of second

DNA strand by DNA

polymerase

Reverse

transcriptase

cDNA strandbeing synthesized

cDNAof genewithoutintrons

2

3

1

5

4

• Another approach is to

– use an automated DNA-synthesizing machine and

– synthesize a gene of interest from scratch.



Figure 12.12

DNA PROFILING AND FORENSIC SCIENCE

• DNA profiling

– can be used to determine if two samples of genetic

material are from a particular individual and

– has rapidly revolutionized the field of forensics,

the scientific analysis of evidence from crime

scenes.

• To produce a DNA profile, scientists compare

sequences in the genome that vary from person to

person.


Figure 12.13-3

DNA isolated

DNA amplified

DNA compared

Crime scene Suspect 1 Suspect 21

2

3

Figure 12.UN02

Crime scene Suspect 1 Suspect 2

DNA

Polymerase chain

reaction (PCR)

amplifies STR

sites

Longer

DNA

fragments

Shorter

DNA

fragments

DNA fragments compared by gel electrophoresis

Gel

(Bands of shorter fragments move faster toward the positive pole.)

Investigating Murder, Paternity, and Ancient DNA

• DNA profiling can be used to

– test the guilt of suspected criminals,

– identify tissue samples of victims,

– resolve paternity cases,

– identify contraband animal products, and

– trace the evolutionary history of organisms.


Figure 12.14

DNA Profiling Techniques The Polymerase Chain Reaction (PCR)

• The polymerase chain reaction (PCR)

– is a technique to copy quickly and precisely a

specific segment of DNA and

– can generate enough DNA, from even minute

amounts of blood or other tissue, to allow DNA

profiling.


Figure 12.15

InitialDNAsegment

Number of DNA molecules

1 2 4 8

Short Tandem Repeat (STR) Analysis

• How do you test if two samples of DNA come from

the same person?

• Repetitive DNA

– makes up much of the DNA that lies between

genes in humans and

– consists of nucleotide sequences that are present

in multiple copies in the genome.


• Short tandem repeats (STRs) are

– short sequences of DNA and

– repeated many times, tandemly (one after

another), in the genome.

• STR analysis

– is a method of DNA profiling and

– compares the lengths of STR sequences at

specific sites in the genome.

Short Tandem Repeat (STR) Analysis


Figure 12.16

Crime scene DNA

Suspect’s DNA

Same number of

short tandem repeats

Different numbers of

short tandem repeats

STR site 1 STR site 2

AGAT

AGAT GATA

GATA

Gel Electrophoresis

• STR analysis

– compares the lengths of DNA fragments and

– uses gel electrophoresis, a method for sorting

macromolecules—usually proteins or nucleic

acids—primarily by their

– electrical charge and

– size.


Figure 12.17-3

Band of longest

(slowest) fragments

Band of shortest

(fastest) fragments

Mixture of DNA

fragments of

different sizes

Power

source

• The DNA fragments are visualized as ―bands‖ on

the gel.

• The differences in the locations of the bands reflect

the different lengths of the DNA fragments.

Gel Electrophoresis


Figure 12.18

Amplifiedcrime sceneDNA

Amplifiedsuspect’sDNA

Longerfragments

Shorterfragments

• Gel electrophoresis may also be used for RFLP

analysis, in which DNA molecules are exposed to

a restriction enzyme, producing fragments that are

compared and made visible by gel electrophoresis.

RFLP Analysis


Figure 12.19Crime scene DNA Suspect’s DNA

Fragment w

Fragment x

Fragment y

Longer

fragments

Shorter

fragments

Fragment z

Fragment y

Crime scene

DNA

Suspect’s

DNA

Cut

Cut Cut

Restriction

enzymes

added

x

wy y

z

GENOMICS AND PROTEOMICS

• Genomics is the study of complete sets of genes

(genomes).

– The first targets of genomics research were

bacteria.

– As of 2011,

– the genomes of more than 1,700 species have

been published and

– more than 8,000 are in progress.


Table 12.1

The Human Genome Project

• Begun in 1990, the Human Genome Project was a

massive scientific endeavor to

– determine the nucleotide sequence of all the DNA in

the human genome and

– identify the location and sequence of every gene.


• At the completion of the project,

– more than 99% of the genome had been

determined to 99.999% accuracy,

– about 3 billion nucleotide pairs were identified,

– about 21,000 genes were found, and

– about 98% of the human DNA was identified as

noncoding.



• The Human Genome Project can help map the

genes for specific diseases such as

– Alzheimer’s disease and

– Parkinson’s disease.



Figure 12.20

Tracking the Anthrax Killer

• In October 2001,

– a Florida man died after inhaling anthrax and

– by the end of the year, four other people had also

died from anthrax.



• In 2008, investigators

– completed a whole-genome analysis of the spores

used in the attack,

– found four unique mutations, and

– traced the mutations to a single flask at an Army

facility.


Figure 12.21

Anthrax

spore

Envelope

containing

anthrax spores

Co

lori

ze

d S

EM


• Although never charged, an army research scientist

suspected in the case committed suicide in 2008,

and the case remains officially unsolved.


• The anthrax investigation is just one example of the new field of bioinformatics, the application of computational tools to molecular biology. Additional examples include

– evidence that a Florida dentist transmitted HIV to several patients,

– tracing the West Nile virus outbreak in 1999 to a single natural strain of virus infecting birds and people, and

– determining that our closest living relative, the chimpanzee (Pan troglodytes), shares 96% of our genome.



Genome-Mapping Techniques

• Genomes are most often sequenced using the

whole-genome shotgun method, in which

– the entire genome is chopped into fragments using

restriction enzymes,

– all the fragments are cloned and sequenced, and

– computers running specialized mapping software

reassemble the millions of overlapping short

sequences into a single continuous sequence for

every chromosome—an entire genome.


Figure 12.22-5

Chromosome

Chop up with

restriction enzyme

Sequence fragments

DNA fragments

Align fragments

Reassemble

full sequence

Figure 12.22a

The Process of Science: Can Genomics Cure Cancer?

• Observation: A few patients responded quite

dramatically to a new drug, gefitinib, which

– targets a protein called EGFR found on the surface

of cells that line the lungs and

– is used to treat lung cancer.

• Question: Are genetic differences among lung

cancer patients responsible for the differences in

gefitinib’s effectiveness?


• Hypothesis: Mutations in the EGFR gene were

causing the different responses to gefitinib.

• Prediction: DNA profiling that focuses on the

EGFR gene would reveal different DNA sequences

in the tumors of responsive patients compared with

the tumors of unresponsive patients.



• Experiment: The EGFR gene was sequenced in

the cells extracted from the tumors of

– five patients who responded to the drug and

– four who did not.



• Results: The results were quite striking.

– All five tumors from gefitinib-responsive patients

had mutations in EGFR.

– None of the other four tumors did.

– These results suggest that doctors can use DNA

profiling techniques to screen lung cancer patients

for those who are most likely to benefit from

treatment with this drug.



Figure 12.23

Proteomics

• Success in genomics has given rise to

proteomics, the systematic study of the full set of

proteins found in organisms.

• To understand the functioning of cells and

organisms, scientists are studying

– when and where proteins are produced and

– how they interact.


HUMAN GENE THERAPY

• Human gene therapy

– is a recombinant DNA procedure,

– seeks to treat disease by altering the genes of the

afflicted person, and

– often replaces or supplements the mutant version

of a gene with a properly functioning one.


Figure 12.UN03

RNA versionof a normalhuman gene

Virus withRNA genome

Bonemarrow

A normal human gene is transcribedand translated in a patient, potentiallycuring the genetic disease permanently

Figure 12.24

Normal

human gene

Healthy person

The engineered

cells are injected

into the patient.

Bone of person

with diseaseBone

marrow

Bone marrow cell from the patient

Viral DNA carrying the human gene

inserts into the cell’s chromosome.

Bone marrow cells of the patient

are infected with the virus.

Inserted human RNA

RNA genome of virus

An RNA version of a normal human

gene is inserted into a harmless

RNA virus.

1

2

3

4

• Severe combined immunodeficiency (SCID) is

– a fatal inherited disease and

– caused by a single defective gene that prevents

the development of the immune system.

• SCID patients quickly die unless treated with

– a bone marrow transplant or

– gene therapy.

HUMAN GENE THERAPY


• From 2000 to 2011, gene therapy has cured 22

children with inborn SCID.

• However, there have been some serious side

effects. Four of the children developed leukemia,

which proved fatal to one.

HUMAN GENE THERAPY


SAFETY AND ETHICAL ISSUES

• As soon as scientists realized the power of DNA

technology, they began to worry about potential

dangers such as the

– creation of hazardous new pathogens and

– transfer of cancer genes into infectious bacteria

and viruses.


SAFETY AND ETHICAL ISSUES

• Strict laboratory safety procedures have been

designed to

– protect researchers from infection by engineered

microbes and

– prevent microbes from accidentally leaving the

laboratory.


Figure 12.25

The Controversy over Genetically Modified Foods

• GM strains account for a significant percentage of several staple crops in the United States.

• Advocates of a cautious approach are concerned that

– crops carrying genes from other species might harm the environment,

– GM foods could be hazardous to human health, and/or

– transgenic plants might pass their genes to close relatives in nearby wild areas.


Figure 12.26

• Negotiators from 130 countries (including the

United States) agreed on a Biosafety Protocol that

– requires exporters to identify GM organisms

present in bulk food shipments and

– allows importing countries to decide whether the

shipments pose environmental or health risks.



• In the United States, all projects are evaluated for

potential risks by a number of regulatory agencies,

including the

– Food and Drug Administration,

– Environmental Protection Agency,

– National Institutes of Health, and

– Department of Agriculture.



Ethical Questions Raised by DNA Technology

• DNA technology raises legal and ethical questions—few of which have clear answers.

– Should genetically engineered human growth hormone be used to stimulate growth in HGH-deficient children?

– Should we try to eliminate genetic defects in our children and their descendants?

– Should people use mail-in kits that can tell healthy people their relative risk of developing various diseases?


Figure 12.27

• DNA technologies raise many complex issues that

have no easy answers.

• We as a society and as individuals must become

educated about DNA technologies to address the

ethical questions raised by their use.

Ethical Questions Raised by DNA Technology


Evolution Connection:The Y Chromosome as a Window on History

• Barring mutations, the human Y chromosome

passes essentially intact from father to son.

• By comparing Y DNA, researchers can learn about

the ancestry of human males.


• DNA profiling of the Y chromosome has revealed

that

– nearly 16 million men currently living may be

descended from Genghis Khan,

– nearly 10% of Irish men were descendants of Niall

of the Nine Hostages, a warlord who lived during

the 1400s, and

– the Lemba people of southern Africa are

descended from ancient Jews.

Evolution Connection:The Y Chromosome as a Window on History


Figure 12.28

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Chapter 12 · Campbell Essential Biology, ... Chapter 12 DNA Technology. Biology and Society: DNA, Guilt, and Innocence ... Figure 12.8 Plasmid Bacterial cell 1 Isolate plasmids