Chapter 12

Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings

PowerPoint Lectures forBiology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon

Lectures by Chris Romero

Chapter 12Chapter 12

DNA Technology and Genomics


DNA and Crime Scene Investigations

• Many violent crimes go unsolved

– lack of enough evidence

• If biological fluids are left at a crime scene

– DNA can be isolated from them


• DNA fingerprinting: set of laboratory procedures

– determines w/near certainty whether 2 samples of DNA are from same individual

– powerful tool for CSIs

Investigator at oneof the crime scenes(above), Narborough,England (left)


BACTERIAL PLASMIDS AND GENE CLONING

12.1 Plasmids are used to customize bacteria: An overview

• Gene cloning is 1 application

• DNA technology

– Methods for studying & manipulating genetic material


• Researchers can insert desired genes into plasmids, creating recombinant DNA

– And insert those plasmids into bacteria

Figure 12.1


Bacterium

Bacterialchromosome

Plasmid

1 Plasmidisolated

3 Gene insertedinto plasmid

2 DNAisolated

Cell containing geneof interest

DNA

Gene ofinterest

Recombinant DNA(plasmid)

4 Plasmid put intobacterial cell

Recombinantbacterium

5 Cell multiplies withgene of interest

Copies of proteinCopies of gene

Clone of cells

Gene for pestResistance inserted into plants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Protein used to make snow form at highertemperature

Figure 12.1


• If the recombinant bacteria multiply into a clone

– The foreign genes are also copied


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


• Creating recombinant DNA using REs & DNA ligase

Restriction enzymerecognition sequence

G A A T T CC T T A A G

DNA1

2

3

4

C T T A A

A AT TC

A AT TCG

C T T A A

Addition of a DNAfragment fromanother source

Two (or more)fragments sticktogether bybase-pairing

G A AT T CC T TA A G

G A AT T CC T TA A G

5

DNA ligasepastes the strand

Restriction enzymecuts the DNA intofragments

Recombinant DNA molecule

G

G

Sticky end

G

Figure 12.2


12.3 Genes can be cloned in recombinant plasmids: A closer look

• Bacteria take the recombinant plasmids from their surroundings

– reproduce

– cloning the plasmids and the genes they carry


• Cloning a gene in a bacterial plasmid

1 Isolate DNAfrom two sources

2 Cut both DNAswith the samerestriction enzyme

E.coli

Plasmid DNA

Gene VSticky ends3 Mix the DNAs;

they join bybase-pairing

4 Add DNA ligaseto bond the DNA covalently

5 Put plasmid into bacteriumby transformation

Gene VRecombinant DNA plasmid

Recombinant bacterium

6 Clone the bacterium

Bacterial clone carrying manycopies 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

– 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

• widely used to produce medicines and to diagnose diseases

CONNECTION


Therapeutic hormones

• In 1982, humulin, human insulin produced by bacteria

– 1st recombinant drug approved by FDA

Figure 12.7A


Diagnosis and Treatment of Disease

• DNA technology

– Is being used increasingly in disease diagnosis


Vaccines

• DNA technology

– Is also helping medical researchers develop vaccines

Figure 12.7B


pGLO LAB


12.10 Gel electrophoresis sorts DNA molecules by size

+ +

– –

Powersource

Gel

Mixture of DNAmolecules ofdifferent sizes

Longermolecules

Shortermolecules

Completed gel

Figure 12.10

RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING


12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences


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

CCGG

GGCC

ACGG

TGCC

CCGG

GGCC

CCGG

GGCC

Figure 12.11A


• After digestion by restriction enzymes

– fragments are run through a gel

–

+

Longerfragments

Shorterfragments

x

w

y

z

y

1 2

Figure 12.11B


Using DNA Probes to Detect Harmful Alleles

• Radioactive probes

– Can reveal DNA bands of interest on a gel


• 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

IIIIII

I

II

IIIFigure 12.11C


12.12 DNA technology is used in courts of law

CONNECTION


• DNA fingerprinting can help solve crimes

Defendant’sblood

Blood fromdefendant’s clothes Victim’s

blood

Figure 12.12A Figure 12.12B


12.13 Gene therapy may someday help treat a variety of diseases

• Gene therapy

– Is the alteration of an afflicted individual’s genes

CONNECTION


Gene Therapy Cloned gene(normal allele)

1 Insert normal geneinto virus

2 Infect bone marrowcell with virus

3 Viral DNA insertsinto chromosome

4 Inject cellsinto patient Bone

marrow

Bone marrowcell from patient

Viral nucleicacid

Retrovirus

Figure 12.13


• Gene therapy

– May one day be used to treat both genetic diseases and nongenetic disorders

• Unfortunately, progress is slow


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 copies for analysis

1 2 4 8

InitialDNAsegment

Number of DNA moleculesFigure 12.14


Figure 12.15

GENOMICS CONNECTION

12.15 The Human Genome Project is an ambitious application of DNA technology

• The Human Genome Project, begun in 1990 and now largely completed

– Genetic and physical mapping of chromosomes, followed by DNA sequencing


• The data are providing insight into

– Development, evolution, and many diseases


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


• Much of the noncoding DNA consists of repetitive nucleotide sequences

– And transposons that can move about within the genome


12.17 The science of genomics compares whole genomes

• The sequencing of many prokaryotic and eukaryotic genomes

CONNECTION


• Besides being interesting themselves...

– Nonhuman genomes can be compared with the human genome

Table 12.17


• Proteomics

– Is the study of the full sets of proteins produced by organisms


12.18 Genetically modified organisms are transforming agriculture

GENETICALLY MODIFIED ORGANISMS: CONNECTION


• Recombinant DNA technology

– Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms

Agrobacterium tumefaciens

DNA containinggene for desired trait

Tiplasmid

1

Insertion of geneinto plasmid usingrestriction enzymeand DNA ligase

RecombinantTi plasmid

2

Introductioninto plantcells inculture

3

Regenerationof plant

Plant with new traitT DNA carrying new

gene within plant chromosome

Plant cell

T DNA

Restriction site

Figure 12.18A


• Transgenic organisms

– have had genes from other organisms inserted into their genomes

Figure 12.18B


• A number of important crops and plants

– Are genetically modified


12.19 Could GM organisms harm human health or the environment?

• Development of GM organisms

– Requires significant safety measures

CONNECTION

Figure 12.19A


• Genetic engineering involves risks

– Such as ecological damage from GM crops

Figure 12.19B


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



12.17 The science of genomics compares whole genomes

• The sequencing of many prokaryotic and eukaryotic genomes

– Has produced data for genomics, the study of whole genomes


12.4 Cloned genes can be stored in genomic libraries

• Genomic libraries, sets of DNA fragments containing all of an organism’s genes

– Can be constructed and stored in cloned bacterial plasmids or phages

Recombinantplasmid

Genome cut up withrestriction enzyme

Recombinantphage DNA

or

Bacterialclone

Phageclone

Phage libraryPlasmid libraryFigure 12.4


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 ofeukaryoticgene

Exon Intron Exon Intron Exon

1 Transcription

2 RNA splicing(removes introns)

3 Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand

4 Breakdown of RNA

5 Synthesis of secondDNA strand

RNA transcript

mRNA

Reverse transcriptase

cDNA strand

cDNA of gene(no introns)

Test tube

Figure 12.5


12.8 Nucleic acid probes identify clones carrying specific genes

• DNA technology methods

– Can be used to identify specific pieces of DNA

RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING


• A nucleic acid probe

– Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA

– Can tag a desired gene in a library

Radioactiveprobe (DNA)

Single-strandedDNA

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Base pairingindicates thegene of interest

A T C C G A

A T G C G C T T A T C G

A G C

C T

T A

T G

C A

T

A T C C

G A

A G G T A G

G C T A A

Figure 12.8


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


• DNA microarray

1 mRNAisolated

Reverse transcriptaseand fluorescent DNAnucleotides

2 cDNA madefrom mRNA

4 UnboundcDNA rinsedaway

3 cDNA appliedto wells

DNA microarray

Each well contains DNAfrom a particular gene

Actual size(6,400 genes)

Nonfluorescentspot

Fluorescentspot

cDNA

DNA of anexpressed gene

DNA of anunexpressed gene

Figure 12.9

Chapter 12

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