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8/13/2019 DNA Technology Genetic Engineering

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Copyright 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

10.22 Bacteria can transfer DNA in three ways

Bacteria can transfer genes from cell to cell byone of three processes

Transformation, transduction, or

conjugationDNA enters

cell

Fragment of DNA

from anotherbacterial cell

Bacterial

chromosome

(DNA)

Phage

F

ragment of

DNA fromanother

bacterial cell

(former phage

host)

Phage

Sex pili

Mating bridge

Donor cell

(male)

Recipient cell

(female)

Figure 10.22AC

Intro to Genetic Engineering


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Once new DNA gets into a bacterial cell

Part of it may then integrate into therecipients chromosome

Recipient cells

chromosome

Recombinant

chromosome

Donated DNACrossovers Degraded DNA

Figure 10.22D


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10.23 Bacterial plasmids can serve as carriers for

gene transfer Plasmids

Are small circular DNA molecules separate

from the bacterial chromosome


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Plasmids can serve as carriers

For the transfer of genes

Plasmids

ColorizedTEM2,0

00

Cell now male

Plasmid completes transfer

and circularizes

F factor starts replication

and transfer

Male (donor) cell

Bacterial chromosome

F factor (plasmid)

Recombination

can occur

Only part of the chromosome

transfers

F factor starts replication

and transfer of chromosome

Origin of F

replication

Bacterialchromosome

Male (donor) cellF factor

(integrated)

Recipient cell

Figure 10.23A

C


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7/53Copyright 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

Researchers can insert desired genes into

plasmids, creating recombinant DNA

And insert those plasmids into bacteriaBacterium

Bacterial

chromosome

Plasmid

1 Plasmid

isolated

3 Gene inserted

into plasmid

2 DNA

isolated

Cell containing gene

of interest

DNAGene of

interest

Recombinant DNA

(plasmid)

4 Plasmid put into

bacterial cell

Recombinant

bacterium

5 Cell multiplies with

gene of interest

Copies of proteinCopies of gene

Clone of cellsGene for pest

resistance

inserted into

plants

Gene used to alter bacteria

for cleaning up toxic wasteProtein used to dissolve blood

clots in heart attack therapy

Protein used to

make snow form

at higher

temperature

Figure 12.1



If the recombinant bacteria multiply into a clone

The foreign genes are also copied

Can insert regulatory sequences to turn

on foreign gene expression in the clone

Can isolate & purify the gene product



12.3 Genes can be cloned in recombinant

plasmids: A closer look Bacteria take the recombinant plasmids from

their surroundings

And reproduce, thereby cloning theplasmids and the genes they carry



Cloning a gene in a bacterial plasmid

1Isolate DNA

from two sources

2Cut both DNAs

with the same

restriction enzyme

E.coli

PlasmidDNA

GeneV

Sticky ends

3 Mix the DNAs;

they join by

base-pairing

4 Add DNA ligase

to bond the DNA covalently

5 Put plasmid into bacterium

by transformation

GeneVRecombinant DNA

plasmid

Recombinant

bacterium

6 Clone the bacterium

Bacterial clone carrying many

copies of the human gene

Human cell

Figure 12.3



CONNECTION

12.6 Recombinant cells and organisms can mass-

produce gene products

Applications of gene cloning include

The mass production of gene products for

medical and other uses

Table 12.6



Different organisms, including bacteria, yeast,

and mammals

Can be used for this purpose

Figure 12.6



12.7 DNA technology is changing the

pharmaceutical industry DNA technology

Is widely used to produce medicines and

to diagnose diseases

CONNECTION


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Therapeutic hormones

In 1982, humulin, human insulin produced bybacteria

Became the first recombinant drug

approved by the Food and DrugAdministration

Figure 12.7A


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Vaccines

DNA technology

Is also helping medical researchers

develop vaccines

Figure 12.7B


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12.8 Nucleic acid probes identify clones carryingspecific genes

DNA technology methods

Can be used to identify specific pieces of

DNA

RESTRICTION FRAGMENT ANALYSIS ANDDNA FINGERPRINTING


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A nucleic acid probe

Is a short, single-stranded molecule ofradioactively labeled or fluorescently

labeled DNA or RNA

Can tag a desired gene in a library

Radioactive

probe (DNA)

Single-stranded

DNA

Mix with single-

stranded DNA from

various bacterial

(or phage) clones

Base pairing

indicates the

gene of interestFigure 12.8


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12.9 DNA microarrays test for the expression of

many genes at once DNA microarray assays

Can reveal patterns of gene expression

in different kinds of cells

CONNECTION


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DNA microarray

1 mRNA

isolated

Reverse transcriptase

and fluorescent DNA

nucleotides

2 cDNA made

from mRNA

4 Unbound

cDNA rinsed

away

3 cDNA applied

to wells

DNA microarray

Each well contains DNA

from a particular gene

Actual size

(6,400 genes)

Nonfluorescent

spotFluorescent

spot

cDNA

DNA of an

expressed geneDNA of an

unexpressed geneFigure 12.9


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12.10 Gel electrophoresis sorts DNA molecules

by size

+ +

Power

source

Gel

Mixture of DNA

molecules of

different sizes

Longer

molecules

Shorter

molecules

Completed gel

Figure 12.10


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12.11 Restriction fragment length polymorphisms

can be used to detect differences in DNA

sequences


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How Restriction Fragments Reflect DNA Sequence

Restriction fragment length polymorphisms (RFLPs)

Reflect differences in the sequences of DNA

samples

Crime scene Suspect

w

x

y y

z

CutCut

Cut

DNA from chromosomes

CC

G

G

GG

C

C

A

C

G

G

T

G

C

C

C

C

G

G

G

G

C

C

C

C

G

G

G

G

C

C

Figure 12.11A


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After digestion by restriction enzymes

The fragments are run through a gel

+

Longer

fragments

Shorter

fragments

x

w

y

z

y

1 2

Figure 12.11B


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Using DNA Probes to Detect Harmful Alleles

Radioactive probes

Can reveal DNA bands of interest on a

gel


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Detecting a harmful allele using restriction

fragment analysis 1

2

3

4

5

Restriction fragment

preparation

Gel electrophoresis

Blotting

Radioactive probe

Detection of radioactivity

(autoradiography)

I II III

I II III

Restriction

fragments

Filter paper

Probe

Radioactive, single-

stranded DNA (probe)

Film

I

II

III

I

II

IIIFigure 12.11C

CONNECTION


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12.12 DNA technology is used in courts of law

CONNECTION


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DNA fingerprinting can help solve crimes

Defendants

blood

Blood from

defendants clothes Victims

blood

Figure 12.12A Figure 12.12B


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DNA and Crime Scene Investigations

Many violent crimes go unsolved

For lack of enough evidence

If biological fluids are left at a crime scene DNA can be isolated from them


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DNA fingerprinting is a set of laboratory

procedures

That determines with near certainty

whether two samples of DNA are from the

same individual

That has provided a powerful tool for

crime scene investigators

Investigator at one

of the crime scenes

(above), Narborough,

England (left)


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12.14 The PCR method is used to amplify DNA

sequences

The polymerase chain reaction (PCR)

Can be used to clone a small sample of

DNA quickly, producing enough copiesfor analysis

1 2 4 8

InitialDNA

segment

Number of DNA moleculesFigure 12.14

CONNECTION


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12.13 Gene therapy may someday help treat a variety of

diseases

Gene therapy

Is the alteration of an afflicted individuals genes

CONNECTION

Cloned gene

(normal allele) 1 Insert normal gene

into virus

2 Infect bone marrow

cell with virus

3 Viral DNA insertsinto chromosome

4 Inject cells

into patient

Bone

marrow

Bone marrowcell from patient

Viral nucleic

acid

Retrovirus

Figure 12.13


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Gene therapy

May one day be used to treat bothgenetic diseases and nongenetic

disorders

Unfortunately, progress is slow

GENOMICS


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Figure 12.15

GENOMICSCONNECTION

12.15 The Human Genome Project is an ambitiousapplication of DNA technology

The Human Genome Project, begun in 1990 and

now largely completed, involved

Genetic and physical mapping of

chromosomes, followed by DNA sequencing


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The data are providing insight into

Development, evolution, and manydiseases

CONNECTION


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12.17 The science of genomics compares whole

genomes

The sequencing of many prokaryotic and

eukaryotic genomes

Has produced data for genomics, thestudy of whole genomes

CONNECTION


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Besides being interesting themselves

Nonhuman genomes can be comparedwith the human genome

Table 12.17


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Proteomics

Is the study of the full sets of proteinsproduced by organisms

GENETICALLY MODIFIED ORGANISMS


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12.18 Genetically modified organisms aretransforming agriculture

GENETICALLY MODIFIED ORGANISMSCONNECTION


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Recombinant DNA technology

Can be used to produce new geneticvarieties of plants and animals,

genetically modified (GM) organisms

Agrobacterium tumefaciens

DNA containing

gene for desired trait

Ti

plasmid

1

Insertion of geneinto plasmid using

restriction enzyme

and DNA ligase

Recombinant

Ti plasmid

2

Introductioninto plant

cells in

culture

3

Regeneration

of plant

Plant with new traitT DNA carrying new

gene within plant chromosome

Plant cell

T DNA

Restriction

siteFigure 12.18A


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Transgenic organisms

Are those that have had genes fromother organisms inserted into their

genomes

Figure 12.18B


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A number of important crops and plants

Are genetically modified

CONNECTION


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12.19 Could GM organisms harm human health

or the environment?

Development of GM organisms

Requires significant safety measures

CONNECTION

Figure 12.19A


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Genetic engineering involves risks

Such as ecological damage from GMcrops

Figure 12.19B

CONNECTION


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12.20 Genomics researcher Eric Lander

discusses the Human Genome Project

Genomics pioneer Eric Lander

Points out that much remains to be

learned from the Human Genome Project

CONNECTION

Figure 12.20


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12.17 The science of genomics compares whole

genomes

The sequencing of many prokaryotic and

eukaryotic genomes

Has produced data for genomics, thestudy of whole genomes


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The following covers material wedid not review during class, but is

covered in the textbook. You are

not responsible for knowing the

following for tests, but it may be

useful for understanding the topic.


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12.4 Cloned genes can be stored in genomic

libraries

Genomic libraries, sets of DNA fragments

containing all of an organisms genes

Can be constructed and stored in cloned

bacterial plasmids or phages

Recombinant

plasmid

Genome cut up with

restriction enzyme

Recombinant

phage DNA

or

Bacterial

clone

Phage

clone

Phage libraryPlasmid libraryFigure 12.4


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12.2 Enzymes are used to cut and paste DNA

The tools used to make recombinant DNA are

Restriction enzymes, which cut DNA at

specific sequences

DNA ligase, which pastes DNA

fragments together


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Creating recombinant DNA using restriction

enzymes and DNA ligaseRestriction enzyme

recognition sequence

G A A T T CC T T A A GDNA1

2

3

4

Addition of a DNA

fragment fromanother source

Two (or more)

fragments stick

together by

base-pairing

G A AT T C

C T TA A GG A AT T C

C T TA A G

5

DNA ligase

pastes the strand

Restriction enzyme

cuts the DNA into

fragments

Recombinant DNA molecule

Sticky end

Figure 12.2


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12.5 Reverse transcriptase helps make genes for cloning

Reverse transcriptase can be used to make smaller,complementary DNA (cDNA) libraries

Containing only the genes that are transcribed

by a particular type of cell

Cell nucleus

DNA of

eukaryotic

gene

Exon Intron Exon Intron Exon

1 Transcription

2 RNA splicing

(removes introns)

3 Isolation of mRNA

from cell and addition

of reverse transcriptase;

synthesis of DNA strand

4 Breakdown of RNA

5 Synthesis of second

DNA strand

RNA

transcript

mRNA

Reverse transcriptase

cDNA strand

cDNA of gene

(no introns)

Test tube

Figure 12.5


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12.16 Most of the human genome does not

consist of genes

The haploid human genome contains about

25,000 genes

And a huge amount of noncoding DNA


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Much of the noncoding DNA consists of

repetitive nucleotide sequences

And transposons that can move about

within the genome

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