HCCS BIOL 1308 Ch 12_Lecture_Presentation

8/11/2019 HCCS BIOL 1308 Ch 12_Lecture_Presentation

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2010 Pearson Education, Inc.

Lectures by Chris C. Romero, updated by Edward J. Zalisko

PowerPoint

Lectures forCampbel l Essent ial Bio logy, Fourth Edition

Eric Simon, Jane Reece, and Jean Dickey

Campbel l Essent ial Biology with Phy sio logy, Third Edition

Eric Simon, Jane Reece, and Jean Dickey

Chapter 12

DNA Technology


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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

Innocent



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DNA technology has led to other advances in the:

Creation of genetically modified crops

Identification and treatment of genetic diseases


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RECOMBINANT DNA TECHNOLOGY

Biotechnology:

Is the manipulation of organisms or their components to make useful

products

Has been used for thousands of years to

Make bread using yeast

Selectively breed livestock for desired traits

Biotechnology today means the use of DNAtechnology, methods

for: Studying and manipulating genetic material

Modifying specific genes

Moving genes between organisms


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Recombinant DNA is formed when scientists combine

nucleotide sequences (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.


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Applications: From Humulin to Foods toPharm 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.


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Making Humulin

In 1982, the worlds first genetically engineered pharmaceutical

product was sold.

Humulin, human insulin:

Was produced by genetically modified bacteria

Was the first recombinant DNA drug approved by the FDA

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.



DNA technology is used to produce medically valuable

molecules, including:

Human growth hormone (HGH)

The hormone EPO, which stimulates production of red blood cells

Vaccines, harmless variants or derivatives of a pathogen used to prevent

infectious diseases



Genetically Modified (GM) Foods

Today, DNA technology is quickly replacing traditional plant-

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 organismcontains a gene from another organism,typically of another species.



In the United States today, roughly one-half of the corn crop and

over three-quarters of the soybean and cotton crops are

genetically modified.

Corn has been genetically modified to resist insect infestation,

such as this damage caused by the European corn borer.



Golden rice has been genetically modified to produce beta-

carotene used in our bodies to make vitamin A.



Pharm Animals

In 2009 the FDA approved the first drug produced by livestock

that has been engineered to carry a human gene.

This product is a human anti-clotting protein collected from goats

milk.


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DNA technology:

May eventually replace traditional animal breeding but

Is not currently used to produce transgenic animals sold as food

Meat may come from livestock that receive genes that produce:

Larger muscles or

Healthy omega-3 fatty acids instead of less healthy fatty acids (already

done in 2006 in pigs)


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Recombinant DNA Techniques

Bacteria are the workhorses of modern biotechnology.

To work with genes in the laboratory, biologists often usebacterial plasmids,small, circular DNA molecules that are

separate from the much larger bacterial chromosome.


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Plasmids

Bacterial

chromosome

Remnant of

bacterium

ColorizedTEM

Figure 12.7


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Plasmids:

Can easily incorporate foreign DNA

Are readily taken up by bacterial cells

Can act as vectors, DNA carriers that move genes from one cell to

another

Are ideal for gene cloning, the production of multiple identical copies ofa gene-carrying piece of DNA


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Recombinant DNA techniques can help biologists produce large

quantities of a desired protein.


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Plasmid

Bacterial cell

Isolateplasmids.

Figure 12.8-1


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Plasmid

Bacterial cell

Isolateplasmids.

DNA

Isolate

DNA.

Cell containingthe gene of interest

Figure 12.8-2


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Plasmid

Bacterial cell

Isolateplasmids.

DNA

Isolate

DNA.

DNA fragments

from cell

Cut both DNAs

with same

enzyme.Gene of

interestOther

genes


Figure 12.8-3


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Plasmid

Bacterial cell

Isolateplasmids.

Gene of interest

Recombinant DNA plasmids

DNA

Isolate

DNA.

DNA fragments

from cell

Cut both DNAs

with same

enzyme.Gene of

interestOther

genes

Mix the DNAs and

join them together.


Figure 12.8-4


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Plasmid

Bacterial cell

Isolateplasmids.

Recombinant bacteria

Gene of interest


Bacteria take up recombinant plasmids.

DNA

Isolate

DNA.

DNA fragments

from cell

Cut both DNAs

with same

enzyme.Gene of

interestOther

genes

Mix the DNAs and

join them together.


Figure 12.8-5


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Plasmid

Bacterial cell

Isolateplasmids.

Clone the bacteria.

Recombinant bacteriaBacterial clone

Gene of interest



DNA

Isolate

DNA.

DNA fragments

from cell

Cut both DNAs

with same

enzyme.Gene of

interestOther

genes

Mix the DNAs and

join them together.


Figure 12.8-6


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Plasmid

Bacterial cell

Isolateplasmids.

Find the clone with

the gene of interest.

Clone the bacteria.


Gene of interest



DNA

Isolate

DNA.

DNA fragments

from cell

Cut both DNAs

with same

enzyme.Gene of

interestOther

genes

Mix the DNAs and

join them together.


Figure 12.8-7


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Plasmid

Bacterial cell

Isolateplasmids.

Some uses

of genes

Gene for pest

resistance

Gene for

toxic-cleanup

bacteria

Genes may be

inserted into

other organisms.

Find the clone with

the gene of interest.

The gene and protein

of interest are isolated

from the bacteria.

Clone the bacteria.


Gene of interest



Harvested

proteins may beused directly.

Some uses

of proteins

Protein for

stone-washing

jeans

DNA


Protein for

dissolving

clots

Isolate

DNA.

DNA fragments

from cell

Cut both DNAs

with same

enzyme.Gene of

interestOther

genes

Mix the DNAs and

join them together.

Figure 12.8-8

A Cl L k C tti d P ti DNA ith


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A Closer Look: Cutting and Pasting DNA withRestriction Enzymes

Recombinant DNA is produced by combining two ingredients:

A bacterial plasmid

The gene of interest

To combine these ingredients, a piece of DNA must be spliced

into a plasmid.



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This splicing process can be accomplished by:

Using restriction enzymes,which cut DNA at specific nucleotide

sequences, and

Producing pieces of DNA called restriction fragmentswith sticky

ends important for joining DNA from different sources

DNA ligase connects the DNA pieces into continuous strands byforming bonds between adjacent nucleotides.

Recognition sequence


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for a restriction enzyme

Restriction

enzyme

DNA

A restriction enzyme cuts

the DNA into fragments.

Figure 12.9-1



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Restriction

enzyme

DNA

A DNA fragment is addedfrom another source.



Figure 12.9-2



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Restriction

enzyme

DNA




Fragments stick together by

base pairing.

Figure 12.9-3



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Restriction

enzyme

DNA

DNA

ligase

Recombinant DNA molecule




Fragments stick together by

base pairing.

DNA ligase joins the

fragments into strands.

Figure 12.9-4

A Closer Look: Obtaining the Gene of Interest


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A Closer Look: Obtaining the Gene of Interest

How can a researcher obtain DNA that encodes a particular gene

of interest?

A shotgun approach yields millions of recombinant plasmids carrying

many different segments of foreign DNA.

A collection of cloned DNA fragments that includes an organisms entire

genome (a complete set of its genes) is called a genomic library.


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Once a genomic library is created, the bacterial clone containing

the desired gene is identified using a specific sequence of

radioactive nucleotides matching those in the desired gene, calleda nucleic acid probe.


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Radioactive probe(single-stranded DNA)

Single-stranded DNA

Mix with single-stranded DNA fromvarious bacterial clones

Base pairing indicates thegene of interest

Figure 12.10


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C ll l


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Cell nucleus

DNA of

eukaryotic

gene

Test tube

Transcription

Exon Intron Exon ExonIntron

Figure 12.11-1

C ll l


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Cell nucleus

DNA of

eukaryotic

gene

RNAtranscript

mRNA

Test tube

Transcription

Introns removed and

exons spliced together


Figure 12.11-2

Cell n cle s


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Cell nucleus

DNA of

eukaryotic

gene

RNAtranscript

mRNA

Test tube

Reverse

transcriptase

Transcription

Introns removed and


Isolation of mRNA from

cell and addition of

reverse transcriptase


Figure 12.11-3

Cell nucleus


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Cell nucleus

DNA of

eukaryotic

gene

RNAtranscript

mRNA

Test tube

Reverse

transcriptase

cDNA strand

being synthesized

Transcription

Introns removed and





Synthesis of cDNA

strand


Figure 12.11-4

Cell nucleus


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Cell nucleus

DNA of

eukaryotic

gene

RNAtranscript

mRNA

Test tube

cDNA of gene

without introns

Reverse

transcriptase

cDNA strand

being synthesized

Transcription

Introns removed and





Synthesis of cDNA

strand

Synthesis of second DNA

strand by DNA polymerase


Figure 12.11-5


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Another approach is to:

Use an automated DNA-synthesizing machine and

Synthesize a gene of interest from scratch


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DNA isolated

Crime scene Suspect 1 Suspect 2

Figure 12.13-1


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DNA isolated

DNA amplified


Figure 12.13-2


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DNA isolated

DNA amplified

DNA compared


Figure 12.13-3


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DNA Profiling Techniques


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DNA Profiling TechniquesThe Polymerase Chain Reaction (PCR)

The polymerase chain reaction(PCR):

Is a technique to copy quickly and precisely any segment of DNA and

Can generate enough DNA, from even minute amounts of blood or other

tissue, to allow DNA profiling



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Short Tandem Repeat (STR) Analysis


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Short Tandem Repeat (STR) Analysis

How do you test if two samples of DNA come from the same

person?

RepetitiveDNA:

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


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Short tandem repeats (STRs)are:

Short sequences of DNA

Repeated many times, tandemly (one after another), in the genome

STR analysis:

Is a method of DNA profiling

Compares the lengths of STR sequences at certain sites in the genome


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Crime scene DNA

Suspects DNA

Same number ofshort tandem repeats

Different numbers ofshort tandem repeats

STR site 1 STR site 2

AGAT

AGAT GATA

GATA

Figure 12.16

Gel Electrophoresis


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p

STR analysis:

Compares the lengths of DNA fragments

Uses gel electrophoresis, a method for sorting macromoleculesusually

proteins or nucleic acidsprimarily by their

Electrical charge

Size


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Mixture of DNA

fragments of

different sizes

Power

source

Gel

Figure 12.17-1


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Mixture of DNA

fragments of

different sizes

Power

source

Gel

Figure 12.17-2


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Mixture of DNA

fragments of

different sizes

Power

source

Gel

Completed gel

Band of

longest

(slowest)

fragments

Band of

shortest

(fastest)

fragments

Figure 12.17-3


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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.


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Amplified

crime scene

DNA

Amplified

suspects

DNA

Longer

fragments

Shorter

fragments

Figure 12.18

RFLP Analysis


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Gel electrophoresis may also be used for RFLP analysis, in

which DNA molecules are exposed to a restriction enzyme, which

produces fragments that are compared and made visible by gelelectrophoresis.

y

Restriction enzymes added


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Crime scene

DNA

Suspects

DNA

Fragment w

Fragment x

Fragment y

Longer

fragments

Shorter

fragments

Fragment z

Fragment y

Crime scene

DNA

Suspects

DNA

Cut

Cut Cut

x

w

y y

z

Figure 12.19

GENOMICS AND PROTEOMICS


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Genomicsis the science of studying complete sets of genes

(genomes).

The first targets of genomics were bacteria.

As of 2009, the genomes of nearly one thousand species have been

published, including:

Bakers yeast

Mice

Fruit flies

Rice


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Table 12.1

The Human Genome Project


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Begun in 1990, the HumanGenomeProject was a massive

scientific endeavor:

To determine the nucleotide sequence of all the DNA in the human genome

and

To identify the location and sequence of every gene


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At the completion of the project in 2004:

Over 99% of the genome had been determined to 99.999% accuracy

3.2 billion nucleotide pairs were identified

About 21,000 genes were found

About 98% of the human DNA was identified as noncoding


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The Human Genome Project can help map the genes for specific

diseases such as:

Alzheimers disease

Parkinsons disease

Tracking the Anthrax Killer


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In October 2001:

A Florida man died after inhaling anthrax

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

Traced the mutations to a single flask at an Army facility

Envelope


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Anthrax

spore

Envelope

containing

anthrax spores

Figure 12.21


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The anthrax investigation is just one example of the new field of

comparative genomics, the comparison of whole genomes.

Comparative genomics has also provided strong evidence that:

A Florida dentist transmitted HIV to several patients

The West Nile virus outbreak in 1999 was a single natural strain of virus

infecting birds and humans Our closest living relative, the chimpanzee (Pantroglodytes), shares 96%

of our genome

Genome-Mapping Techniques


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Genomes are most often sequenced using the whole-genome

shotgun method in which:

The entire genome is chopped into fragments using restriction enzymes

The fragments are cloned and sequenced

Computers running specialized mapping software reassemble the millions

of overlapping short sequences into a single continuous sequence for

every chromosomean entire genome


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Chromosome

Chop up with

restriction enzyme

DNA fragments

Figure 12.22-2


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Chromosome

Chop up with

restriction enzyme

Sequencefragments

DNA fragments

Figure 12.22-3


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Chromosome

Chop up with

restriction enzyme

Sequencefragments

DNA fragments

Align

fragments

Figure 12.22-4


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Chromosome

Chop up with

restriction enzyme

Sequencefragments

DNA fragments

Align

fragments

Reassemble

full sequence

Figure 12.22-5


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Begun in 2006, the Human Variome Project:

Seeks to collect information on all of the genetic variations that affect

human health

The Process of Science:C G i C C ?


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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

Is used to treat lung cancer

Question: Are genetic differences among lung cancer patients

responsible for the differences in gefitinibs effectiveness?



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Hypothesis: Mutations in the EGFRgene were causing the

different responses to gefitinib.

Prediction: DNA profiling that focuses on the EGFRgene would

reveal different DNA sequences in the tumors of responsive

patients compared with the tumors of unresponsive patients.


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Experiment: The EGFRgene was sequenced in the cells

extracted from the tumors of:

Five patients who responded to the drug

Four who did not

Results: The results were quite striking.

All five tumors from gefitinib-responsive patients had mutations inEGFR.

None of the other four tumors did.


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Figure 12.23

Proteomics


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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


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Human gene therapy:

Is a recombinant DNA procedure

Seeks to treat disease by altering the genes of the afflicted person

Often replaces or supplements the mutant version of a gene with a

properly functioning one


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Normal human

gene isolated

and cloned

Healthy person

Figure 12.24-1


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Normal human

gene isolated

and cloned

Normal human

gene inserted

into virus

Healthy person

Harmless

virus (vector)

Virus containing

normal human gene

Figure 12.24-2


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Normal human

gene isolated

and cloned

Normal human

gene inserted

into virus

Virus injected

into patient with

abnormal gene

Healthy person

Harmless

virus (vector)

Virus containing

normal human gene

Bonemarrow

Bone of person

with disease

Figure 12.24-3


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SCID is a fatal inherited disease 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


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Since the year 2000, gene therapy has:

Cured 22 children with inborn SCID but

Unfortunately, caused four of the patients to develop leukemia, killing

one of these children

SAFETY AND ETHICAL ISSUES


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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

Transfer of cancer genes into infectious bacteria and viruses


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Strict laboratory safety procedures have been designed to:

Protect researchers from infection by engineered microbes

Prevent microbes from accidentally leaving the laboratory


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Figure 12.25

The Controversy over Genetically ModifiedFoods


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Foods

GM strains account for a significant percentage of several

agricultural crops in the United States.


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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

Transgenic plants might pass their genes to close relatives in nearby wild

areas


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In 2000, negotiators from 130 countries (including the United

States) agreed on a Biosafety Protocol that:

Requires exporters to identify GM organisms present in bulk foodshipments

h i d ll j l d f i l i k


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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

Department of Agriculture

Ethical Questions Raised by DNA Technology

DNA h l i l l d hi l i f f hi h


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DNA technology raises legal and ethical questionsfew of whichhave clear answers.

Should genetically engineered human growth hormone be used tostimulate growth in HGH-deficient children?

Do we have any right to alter an organisms genesor to create neworganisms?

Should we try to eliminate genetic defects in our children and theirdescendants?

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


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Figure 12.27

DNA h l i i l i h h


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DNA technologies raise many complex issues that have no easy

answers.

We as a society and as individuals must become educated aboutDNA technologies to address the ethical questions raised by their

use.

Evolution Connection:Profiling the Y Chromosome


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Profiling the Y Chromosome

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 fili f th Y h h l d th t


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DNA profiling of the Y chromosome has revealed that:

Nearly 10% of Irish men were descendants of Niall of the Nine Hostages,

a warlord who lived during the 5th century

The Lemba people of southern Africa are descended from ancient Jews

8% of males currently living in central Asia may be descended from

Genghis Khan

HCCS BIOL 1308 Ch 12_Lecture_Presentation

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