20 lectures ppt

8/7/2019 20 Lectures PPT

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Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

PowerPoint Lectures for

Biology, Seventh Edition

Neil Campbell and Jane Reece

Lectures by Chris Romero

Chapter 20Chapter 20

DNA Technology

and Genomics


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Overview: Understanding and ManipulatingGenomes

Sequencing of the human genome was largelycompleted by 2003

DNA sequencing has depended on advances in

technology, starting with making recombinant DNA

In recombinant DNA, nucleotide sequences from

two different sources, often two species, are

combined in vitro into the same DNA molecule

Methods for making recombinant DNA are central

to genetic engineering, the direct manipulation of

genes for practical purposes


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DNA technology has revolutionized biotechnology,the manipulation of organisms or their genetic

components to make useful products

An example ofDNA technology is the microarray,

a measurement of gene expression of thousands

of different genes


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Concept 20.1: DNA cloning permits production ofmultiple copies of a specific gene or other DNA segment

To work directly with specific genes, scientists

prepare gene-sized pieces ofDNA in identical

copies, a process called gene cloning


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DNA Cloning and Its Applications: A Preview

Most methods for cloning pieces ofDNA in thelaboratory share general features, such as the use

of bacteria and their plasmids

Cloned genes are useful for making copies of a

particular gene and producing a gene product


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LE 20-2Bacterium

Bacterial

chromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the cloned

gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up toxic waste

Protein dissolves

blood clots in heart

attack therapy

Human growth hor-

mone treats stunted

growth


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Using Restriction Enzymes to Make RecombinantDNA

Bacterial restriction enzymes cut DNA moleculesat DNA sequences called restriction sites

A restriction enzyme usually makes many cuts,

yielding restriction fragments

The most useful restriction enzymes cut DNA in a

staggered way, producing fragments with sticky

ends that bond with complementary sticky ends

of other fragments

DNA ligase is an enzyme that seals the bonds

between restriction fragments


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LE 20-3Restriction site

DNA5d3d

3d5d

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.

One possible combination

DNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.

Fragment from differentDNA molecule cut by thesame restriction enzyme

DNA ligaseseals the strands.

Recombinant DNA molecule

Sticky end


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Animation: Restriction EnzymesAnimation: Restriction Enzymes


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Cloning a Eukaryotic Gene in a Bacterial Plasmid

In gene cloning, the original plasmid is called acloning vector

A cloning vector is a DNA molecule that can carry

foreign DNA into a cell and replicate there


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Producing Clones of Cells

Cloning a human gene in a bacterial plasmid canbe divided into six steps:

1. Vector and gene-source DNA are isolated

2. DNA is inserted into the vector

3. Human DNA fragments are mixed with cutplasmids, and base-pairing takes place

4. Recombinant plasmids are mixed with bacteria

5. The bacteria are plated and incubated

6. Cell clones with the right gene are identified

Animation: Cloning a GeneAnimation: Cloning a Gene


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LE 20-4_1

Isolate plasmid DNA

and human DNA.

Cut both DNA samples with

the same restriction enzyme.

Mix the DNAs; they join by base pairing.

The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene

(lactose

breakdown)

Human

cell

Restriction

site

ampRgene

(ampicillinresistance)

Bacterial

plasmid Gene of

interest

Sticky

endsHuman DNA

fragments

Recombinant DNA plasmids


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LE 20-4_2

Isolate plasmid DNA

and human DNA.




The products are recombinant plasmids

and many nonrecombinant plasmids.


(lactose

breakdown)

Human

cell

Restriction

site

ampRgene

(ampicillin

resistance)

Bacterial

plasmid Gene of

interest

Sticky

endsHuman DNA

fragments


Introduce the DNA into bacterial cells

that have a mutation in their own lacZ

gene.

Recombinant

bacteria


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LE 20-4_3

Isolate plasmid DNA

and human DNA.




The products are recombinant plasmids

and many nonrecombinant plasmids.


(lactose

breakdown)

Human

cell

Restriction

site

ampRgene

(ampicillinresistance)

Bacterial

plasmid Gene ofinterest

Sticky

endsHuman DNAfragments


Introduce the DNA into bacterial cells

that have a mutation in their own lacZ

gene.

Recombinant

bacteria

Plate the bacteria on agar

containing ampicillin and X-gal.

Incubate until colonies grow.

Colony carrying non-

recombinant plasmid

with intact lacZgene

Colony carrying

recombinant

plasmid with

disrupted lacZgene

Bacterial

clone


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Identifying Clones Carrying a Gene ofInterest

A clone carrying the gene of interest can beidentified with a nucleic acid probe having a

sequence complementary to the gene

This process is called nucleic acid hybridization

An essential step in this process is denaturation of

the cells DNA, separation of its two strands


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LE 20-5

Master plate

Filter

Solution

containing

probe

Filter liftedand flipped over

Radioactive

single-stranded

DNA

Probe

DNA

Gene of

interest

Single-stranded

DNA from cell

Film

Hybridization

on filter

Master plate

Colonies

containing

gene of

interest

A special filter paper

is pressed against

the master plate,

transferring cells to

the bottom side ofthe filter.

The filter is treated to break

open the cells and denature

their DNA; the resulting

single-stranded DNA

molecules are treated so thatthey stick to the filter.

The filter is laid

under photographic

film, allowing any

radioactive areas to

expose the film(autoradiography).

After the

developed film is

flipped over, the

reference marks

on the film andmaster plate are

aligned to locate

colonies carrying

the gene of

interest.


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Storing Cloned Genes in DNA Libraries

A genomic library that is made using bacteria isthe collection of recombinant vector clones

produced by cloning DNA fragments from an

entire genome

A genomic library that is made using

bacteriophages is stored as a collection of phage

clones


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LE 20-6

Bacterialclones RecombinantplasmidsRecombinant

phage DNA

or

Foreign genomecut up with

restriction

enzyme

Phage

clones

Plasmid library Phage library


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A complementary DNA (cDNA) library is made bycloning DNA made in vitro by reverse transcription

of all the mRNA produced by a particular cell

A cDNA library represents only part of thegenomeonly the subset of genes transcribed

into mRNA in the original cells


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Cloning and Expressing Eukaryotic Genes

As an alternative to screening a DNA library,clones can sometimes be screened for a desired

gene based on detection of its encoded protein

After a gene has been cloned, its protein productcan be produced in larger amounts for research


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Bacterial Expression Systems

Several technical difficulties hinder expression ofcloned eukaryotic genes in bacterial host cells

To overcome differences in promoters and other

DNA control sequences, scientists usually employ

an expression vector, a cloning vector that

contains a highly active prokaryotic promoter


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Eukaryotic Cloning and Expression Systems

The use of cultured eukaryotic cells as host cellsand yeast artificial chromosomes (YACs) as

vectors helps avoid gene expression problems

YACs behave normally in mitosis and can carry

more DNA than a plasmid

Eukaryotic hosts can provide the posttranslationalmodifications that many proteins require


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One method of introducing recombinant DNA intoeukaryotic cells is electroporation, applying a brief

electrical pulse to create temporary holes in

plasma membranes

Alternatively, scientists can inject DNA into cells

using microscopic needles

Once inside the cell, the DNA is incorporated into

the cells DNA by natural genetic recombination


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Amplifying DNA in Vitro: The Polymerase ChainReaction (PCR)

The polymerase chain reaction, PCR, can producemany copies of a specific target segment ofDNA

A three-step cycleheating, cooling, andreplicationbrings about a chain reaction that

produces an exponentially growing population ofidentical DNA molecules


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LE 20-7

Genomic DNA

Target

sequence

5d

3d

3d

5d

5d

3d

3d

5d

Primers

Denaturation:

Heat brieflyto separate DNAstrands

Annealing:Cool to allowprimers to formhydrogen bondswith ends oftarget sequence

Extension:DNA polymeraseadds nucleotides tothe 3d end of eachprimer

Cycle 1yields

2molecules

New

nucleo-

tides

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence


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Concept 20.2: Restriction fragment analysisdetects DNA differences that affect restriction sites

Restriction fragment analysis detects differencesin the nucleotide sequences ofDNA molecules

Such analysis can rapidly provide comparative

information about DNA sequences


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Gel Electrophoresis and Southern Blotting

One indirect method of rapidly analyzing andcomparing genomes is gel electrophoresis

This technique uses a gel as a molecular sieve to

separate nuclei acids or proteins by size

Video: Biotechnology LabVideo: Biotechnology Lab


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LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmolecules

of differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules


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In restriction fragment analysis, DNA fragmentsproduced by restriction enzyme digestion of a

DNA molecule are sorted by gel electrophoresis

Restriction fragment analysis is useful forcomparing two different DNA molecules, such as

two alleles for a gene


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LE 20-9Normal F-globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant F-globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles ofF-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp

175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles


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A technique called Southern blotting combines gelelectrophoresis with nucleic acid hybridization

Specific DNA fragments can be identified by

Southern blotting, using labeled probes that

hybridize to the DNA immobilized on a blot of gel


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LE 20-10

DNA + restriction enzyme Restrictionfragments

- NormalF-globinallele

-- Sickle-cellallele

--- Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

- -- --- Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

- -- ---

Radioactivelylabeled probe

forF-globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant F-globin

Fragment fromsickle-cellF-globin allele

Fragment fromnormal F-globinallele

Autoradiography.

- -- ---

Film overpaper blot


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Restriction Fragment Length Differences asGenetic Markers

Restriction fragment length polymorphisms(RFLPs, or Rif-lips) are differences in DNA

sequences on homologous chromosomes that

result in restriction fragments of different lengths

A RFLP can serve as a genetic marker for aparticular location (locus) in the genome

RFLPs are detected by Southern blotting


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Concept 20.3: Entire genomes can be mapped atthe DNA level

The most ambitious mapping project to date hasbeen the sequencing of the human genome

Officially begun as the Human Genome Project in

1990, the sequencing was largely completed by2003

Scientists have also sequenced genomes of other

organisms, providing insights of general biological

significance


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Genetic (Linkage) Mapping: Relative Ordering ofMarkers

The first stage in mapping a large genome isconstructing a linkage map of several thousand

genetic markers throughout each chromosome

The order of markers and relative distances

between them are based on recombination

frequencies


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LE 20-11

Cytogenetic map

Genes located

by FISH

Chromosome

bands

Genetic

markers

Genetic (linkage)

mapping

Physical mapping

Overlapping

fragments

DNA sequencing


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Physical Mapping: Ordering DNA Fragments

A physical map is constructed by cutting a DNAmolecule into many short fragments and arranging

them in order by identifying overlaps

Physical mapping gives the actual distance in

base pairs between markers


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DNA Sequencing

Relatively short DNA fragments can be sequencedby the dideoxy chain-termination method

Inclusion of special dideoxyribonucleotides in the

reaction mix ensures that fragments of various

lengths will be synthesized

E 20 12


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LE 20-12

DNA(template strand)

5d

3d

Primer3d

5d

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3d

5dDNA (templatestrand)

Labeled strands3d

Directionof movementof strands

Laser Detector


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Linkage mapping, physical mapping, and DNAsequencing represent the overarching strategy of

the Human Genome Project

An alternative approach to sequencing genomes

starts with sequencing random DNA fragments

Computer programs then assemble overlapping

short sequences into one continuous sequence

LE 20 13


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LE 20-13

Cut the DNA from

many copies of an

entire chromosome

into overlapping frag-

ments short enough

for sequencing

Clone the fragments

in plasmid or phage

vectors

Sequence each fragment

Order the

sequences into one

overall sequence

with computer

software


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Concept 20.4: Genome sequences provide clues toimportant biological questions

In genomics, scientists study whole sets of genesand their interactions

Genomics is yielding new insights into genome

organization, regulation of gene expression,

growth and development, and evolution


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Identifying Protein-Coding Genes in DNASequences

Computer analysis of genome sequences helpsidentify sequences likely to encode proteins

The human genome contains about 25,000 genes,

but the number of human proteins is much larger

Comparison of sequences of new genes with

those of known genes in other species may help

identify new genes


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Determining Gene Function

One way to determine function is to disable thegene and observe the consequences

Using in vitro mutagenesis, mutations areintroduced into a cloned gene, altering or

destroying its function When the mutated gene is returned to the cell, the

normal genes function might be determined byexamining the mutants phenotype

In nonmammalian organisms, a simpler and fastermethod, RNA interference (RNAi), has been usedto silence expression of selected genes


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Studying Expression of Interacting Groups ofGenes

Automation has allowed scientists to measureexpression of thousands of genes at one timeusing DNA microarray assays

DNA microarray assays compare patterns of gene

expression in different tissues, at different times,or under different conditions

LE 20-14


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LE 20-14

Make cDNA by reverse

transcription, using

fluorescently labeled

nucleotides.

Apply the cDNA mixture to amicroarray, a microscope slide

on which copies of single-

stranded DNA fragments from

the organisms genes are fixed,

a different gene in each spot.

The cDNA hybridizes with any

complementary DNA on the

microarray.

Rinse off excess cDNA; scan

microarray for fluorescent.

Each fluorescent spot

(yellow) represents a gene

expressed in the tissue

sample.

Isolate mRNA.Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNA

microarray

Size of an actual

DNA microarray

with all the genes

of yeast (6,400 spots)


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Comparing Genomes of Different Species

Comparative studies of genomes from related andwidely divergent species provide information in

many fields of biology

The more similar the nucleotide sequences

between two species, the more closely relatedthese species are in their evolutionary history

Comparative genome studies confirm therelevance of research on simpler organisms to

understanding human biology


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Future Directions in Genomics

Genomics is the study of entire genomes

Proteomics is the systematic study of all proteins

encoded by a genome

Single nucleotide polymorphisms (SNPs) provide

markers for studying human genetic variation


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Concept 20.5: The practical applications of DNAtechnology affect our lives in many ways

Many fields benefit from DNA technology andgenetic engineering

i i i


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Medical Applications

One benefit ofDNA technology is identification ofhuman genes in which mutation plays a role in

genetic diseases

Di i f Di


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Diagnosis ofDiseases

Scientists can diagnose many human geneticdisorders by using PCR and primers

corresponding to cloned disease genes, then

sequencing the amplified product to look for the

disease-causing mutation Even when a disease gene has not been cloned,

presence of an abnormal allele can be diagnosed

if a closely linked RFLP marker has been found

LE 20-15


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LE 20 15

DNA

RFLP marker

Disease-causing

allele

Normal allele

Restriction

sites

H G Th


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Human Gene Therapy

Gene therapy is the alteration of an afflictedindividuals genes

Gene therapy holds great potential for treating

disorders traceable to a single defective gene

Vectors are used for delivery of genes into cells

Gene therapy raises ethical questions, such aswhether human germ-line cells should be treated

to correct the defect in future generations

LE 20-16


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Cloned gene

Retrovirus

capsid

Bone

marrow

cell from

patient

Inject engineered

cells into patient.

Insert RNA version of normal allele

into retrovirus.

Viral RNA

Let retrovirus infect bone marrow cells

that have been removed from the

patient and cultured.

Viral DNA carrying the normal

allele inserts into chromosome.

Bone

marrow

Ph ti l P d t


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Pharmaceutical Products

Some pharmaceutical applications ofDNAtechnology:

Large-scale production of human hormones

and other proteins with therapeutic uses

Production of safer vaccines

F i E id


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Forensic Evidence

DNA fingerprints obtained by analysis of tissueor body fluids can provide evidence in criminal and

paternity cases

ADNA fingerprint is a specific pattern of bands of

RFLP markers on a gel

The probability that two people who are notidentical twins have the same DNA fingerprint is

very small Exact probability depends on the number of

markers and their frequency in the population

LE 20-17


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Defendants

blood (D)

Blood from defendants

clothes

Victims

blood (V)

E i t l Cl


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Environmental Cleanup

Genetic engineering can be used to modify themetabolism of microorganisms

Some modified microorganisms can be used to

extract minerals from the environment or degrade

potentially toxic waste materials

Agric lt ral Applications


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Agricultural Applications

DNA technology is being used to improveagricultural productivity and food quality

Animal Husbandry and Pharm Animals


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AnimalHusbandry and PharmAnimals

Transgenic organisms are made by introducinggenes from one species into the genome ofanother organism

Transgenic animals may be created to exploit the

attributes of new genes (such as genes for fastergrowth or larger muscles)

Other transgenic organisms are pharmaceuticalfactories, producers of large amounts of

otherwise rare substances for medical use


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Genetic Engineering in Plants


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Genetic Engineering in Plants

Agricultural scientists have endowed a number of

crop plants with genes for desirable traits

The Ti plasmid is the most commonly used vector

for introducing new genes into plant cells

LE 20-19A b t i t f i


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Agrobacterium tumefaciens

Ti

plasmid

Site where

restriction

enzyme cuts

DNA with

the gene

of interest

T DNA

Recombinant

Ti plasmid

Plant with

new trait

Safety and Ethical Questions Raised by DNA


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Safety and Ethical Questions Raised by DNATechnology

Potential benefits of genetic engineering must be

weighed against potential hazards of creating

harmful products or procedures

Most public concern about possible hazards

centers on genetically modified (GM) organismsused as food

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