Chapter 123.6+DNA...Campbell Biology: Concepts & Connections, Seventh Edition Reece, Taylor, Simon,...

© 2012 Pearson Education, Inc. Lecture by Edward J. Zalisko

PowerPoint Lectures forCampbell Biology: Concepts & Connections, Seventh EditionReece, Taylor, Simon, and Dickey

Chapter 12 DNA Technology and Genomics

▪ DNA technology– has rapidly revolutionized the field of forensics,

– permits the use of gene cloning to produce medical and industrial products,

– allows for the development of genetically modified organisms for agriculture,

– permits the investigation of historical questions about human family and evolutionary relationships, and

– is invaluable in many areas of biological research.

Introduction

© 2012 Pearson Education, Inc.

Figure 12.0_1Chapter 12: Big Ideas

Gene Cloning

DNA Profiling

Genetically ModifiedOrganisms

Genomics

Figure 12.0_2

GENE CLONING


12.1 Genes can be cloned in recombinant plasmids

▪ Biotechnology is the manipulation of organisms or their components to make useful products.

▪ For thousands of years, humans have– used microbes to make wine and cheese and

– selectively bred stock, dogs, and other animals.

▪ DNA technology is the set of modern techniques used to study and manipulate genetic material.


Figure 12.1A


▪ Genetic engineering involves manipulating genes for practical purposes.– Gene cloning leads to the production of multiple,

identical copies of a gene-carrying piece of DNA.

– Recombinant DNA is formed by joining nucleotide sequences from two different sources.– One source contains the gene that will be cloned.

– Another source is a gene carrier, called a vector.– Plasmids (small, circular DNA molecules independent of the

bacterial chromosome) are often used as vectors.


▪ Steps in cloning a gene1. Plasmid DNA is isolated.

2. DNA containing the gene of interest is isolated.

3. Plasmid DNA is treated with a restriction enzyme that cuts in one place, opening the circle.

4. DNA with the target gene is treated with the same enzyme and many fragments are produced.

5. Plasmid and target DNA are mixed and associate with each other.



6. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together.

7. The recombinant plasmid containing the target gene is taken up by a bacterial cell.

8. The bacterial cell reproduces to form a clone, a group of genetically identical cells descended from a single ancestral cell.




Animation: Cloning a GeneRight click on animation / Click play

Figure 12.1B E. coli bacterium

Bacterialchromosome

A plasmidis isolated.

Gene ofinterest

The plasmid is cutwith an enzyme.

Plasmid

The cell’s DNAis isolated.

The cell’s DNA is cutwith the same enzyme.

DNA

Examples of gene use

A cell with DNAcontaining the geneof interest

Geneof interest

The targeted fragmentand plasmid DNAare combined.

DNA ligase is added,which joins the twoDNA molecules.

Geneof interest

Genes may be insertedinto other organisms.

The recombinant plasmidis taken up by a bacteriumthrough transformation.

Examples of protein use

Harvestedproteinsmay beuseddirectly.

The bacteriumreproduces.

Cloneof cells

Recombinantbacterium

RecombinantDNAplasmid

1

3

5

4

2

6

7

9

8

Figure 12.1B_s1

E. colibacterium

Bacterialchromosome


Gene ofinterest

Plasmid


DNA


12

Figure 12.1B_s2

E. colibacterium

Bacterialchromosome


Gene ofinterest

Plasmid


DNA


1

3

2

4



Geneof interest

Figure 12.1B_s3

E. colibacterium

Bacterialchromosome


Gene ofinterest

Plasmid


DNA


1

3

2

4

5



Geneof interest


Figure 12.1B_s4

E. colibacterium

Bacterialchromosome


Gene ofinterest

Plasmid


DNA


1

3

2

4

5

6



Geneof interest


DNA ligase is added,which joins the twoDNA molecules.

Geneof interest


Figure 12.1B_s5

Geneof interest




7

Figure 12.1B_s6

Geneof interest



Cloneof cells



7

8

Figure 12.1B_s7

Geneof interest


Harvestedproteinsmay beuseddirectly.


Cloneof cells



Genes may be insertedinto other organisms.

9

7

8

12.2 Enzymes are used to “cut and paste” DNA

▪ Restriction enzymes cut DNA at specific sequences.– Each enzyme binds to DNA at a different restriction

site.

– Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends.”

– Fragments with complementary sticky ends can associate with each other, forming recombinant DNA.

▪ DNA ligase joins DNA fragments together.



Animation: Restriction EnzymesRight click on animation / Click play

Figure 12.2_s1

A restrictionenzyme cutsthe DNA intofragments.

Restriction enzymerecognition sequence

Restrictionenzyme

Sticky

endStick

yend

DNA1

2

Figure 12.2_s2



Restrictionenzyme

Gene ofinterestA DNA fragment

from anothersource is added.

Sticky

endStick

yend

DNA1

2

3

Figure 12.2_s3



Restrictionenzyme



Two (or more)fragments sticktogether bybase pairing.

Sticky

endStick

yend

DNA1

2

4

3

Figure 12.2_s4



Restrictionenzyme



Two (or more)fragments sticktogether bybase pairing.

Sticky

endStick

yend

DNA ligaseDNA ligasepastes thestrands together.

RecombinantDNA molecule

DNA1

2

4

5

3

12.3 Cloned genes can be stored in genomic libraries

▪ A genomic library is a collection of all of the cloned DNA fragments from a target genome.

▪ Genomic libraries can be constructed with different types of vectors:– plasmid library: genomic DNA is carried by plasmids,

– bacteriophage (phage) library: genomic DNA is incorporated into bacteriophage DNA,

– bacterial artificial chromosome (BAC) library: specialized plasmids that can carry large DNA sequences.


Figure 12.3

A genome is cut up witha restriction enzyme

Recombinantphage DNA

Recombinantplasmid

Bacterialclone

Phageclone

or

Plasmid library Phage library

12.4 Reverse transcriptase can help make genes for cloning

▪ Complementary DNA (cDNA) can be used to clone eukaryotic genes.– In this process, mRNA from a specific cell type is the

template.– Reverse transcriptase produces a DNA strand from

mRNA.– DNA polymerase produces the second DNA strand.


12.4 Reverse transcriptase can help make genes for cloning

▪ Advantages of cloning with cDNA include the ability to– study genes responsible for specialized characteristics

of a particular cell type and– obtain gene sequences

– that are smaller in size,– easier to handle, and– do not have introns.


Figure 12.4

CELL NUCLEUS

DNA of aeukaryoticgene

RNAtranscript

mRNA

TEST TUBEReverse transcriptase

cDNA strandbeing synthesized

Directionof synthesis

Breakdown of RNA

Synthesis of secondDNA strand

Isolation of mRNA fromthe cell and the additionof reverse transcriptase;synthesis of a DNA strand

cDNA of gene(no introns)

Exon Exon ExonIntron Intron

Transcription

RNA splicing (removesintrons and joins exons)

1

2

3

4

5

Figure 12.4_1

CELL NUCLEUS

DNA of aeukaryoticgene

RNAtranscript

mRNA

Exon Intron

Transcription

RNA splicing (removesintrons and joins exons)

1

2

Exon Intron Exon

Figure 12.4_2

TEST TUBEReverse transcriptase

cDNA strandbeing synthesized

Directionof synthesis

Breakdown of RNA

Synthesis of secondDNA strand

Isolation of mRNA fromthe cell and the additionof reverse transcriptase;synthesis of a DNA strand

cDNA of gene(no introns)

3

4

5

12.5 Nucleic acid probes identify clones carrying specific genes

▪ Nucleic acid probes bind very selectively to cloned DNA.– Probes can be DNA or RNA sequences

complementary to a portion of the gene of interest.

– A probe binds to a gene of interest by base pairing.

– Probes are labeled with a radioactive isotope or fluorescent tag for detection.


12.5 Nucleic acid probes identify clones carrying specific genes

▪ One way to screen a gene library is as follows:1. Bacterial clones are transferred to filter paper.

2. Cells are broken apart and the DNA is separated into single strands.

3. A probe solution is added and any bacterial colonies carrying the gene of interest will be tagged on the filter paper.

4. The clone carrying the gene of interest is grown for further study.


Figure 12.5

Radioactivenucleic acid probe(single-stranded

DNA)

Base pairinghighlights thegene of interest.

The probe is mixed withsingle-stranded DNAfrom a genomic library.

Single-strandedDNA

GENETICALLY MODIFIED ORGANISMS


12.6 Recombinant cells and organisms can mass-produce gene products

▪ Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins.

▪ Bacteria are often the best organisms for manufacturing a protein product because bacteria– have plasmids and phages available for use as gene-

cloning vectors,– can be grown rapidly and cheaply,– can be engineered to produce large amounts of a

particular protein, and– often secrete the proteins directly into their growth

medium.



▪ Yeast cells– are eukaryotes,– have long been used to make bread and beer,– can take up foreign DNA and integrate it into their

genomes,– have plasmids that can be used as gene vectors, and– are often better than bacteria at synthesizing and

secreting eukaryotic proteins.



▪ Mammalian cells must be used to produce proteins with chains of sugars. Examples include– human erythropoietin (EPO), which stimulates the

production of red blood cells,

– factor VIII to treat hemophilia, and

– tissue plasminogen activator (TPA) used to treat heart attacks and strokes.


Table 12.6

Table 12.6_1

Table 12.6_2


▪ Pharmaceutical researchers are currently exploring the mass production of gene products by– whole animals or

– plants.

▪ Recombinant animals– are difficult and costly to produce and

– must be cloned to produce more animals with the same traits.


Figure 12.6A

A GOAT CARRYING A GENE FOR A HUMAN BLOOD PROTEIN THAT IS SECRETED IN THE MILK

Figure 12.6B

A PIG THAT HAS BEEN GENETICALLY MODIFIED TO PRODUCE A USEFUL HUMAN PROTEIN

12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine

▪ Products of DNA technology are already in use.– Therapeutic hormones produced by DNA technology

include– insulin to treat diabetes and– human growth hormone to treat dwarfism.

– DNA technology is used to– test for inherited diseases,– detect infectious agents such as HIV, and– produce vaccines, harmless variants (mutants) or derivatives

of a pathogen that stimulate the immune system.


Figure 12.7A

HUMAN INSULIN PRODUCED BY BACTERIA

Figure 12.7B

EQUIPMENT USED IN THE PRODUCTION OF A VACCINE AGAINST HEPATITIS B

12.8 CONNECTION: Genetically modified organisms are transforming agriculture

▪ Genetically modified (GM) organisms contain one or more genes introduced by artificial means.

▪ Transgenic organisms contain at least one gene from another species.



▪ The most common vector used to introduce new genes into plant cells is– a plasmid from the soil bacterium Agrobacterium

tumefaciens and– called the Ti plasmid.


Figure 12.8A_s1

Restrictionsite

The gene isinserted intothe plasmid.

RecombinantTi plasmid

DNA containing thegene for a desired trait

1Ti

plasmid

Agrobacteriumtumefaciens

Figure 12.8A_s2

Restrictionsite


The recombinantplasmid isintroduced intoa plant cell. DNA carrying

the new gene


Plant cellDNA containing thegene for a desired trait

21Ti

plasmid


Figure 12.8A_s3

Restrictionsite


The recombinantplasmid isintroduced intoa plant cell.

The plant cellgrows intoa plant.

DNA carryingthe new gene

A plantwith thenew trait


Plant cellDNA containing thegene for a desired trait

3

21Ti

plasmid



▪ GM plants are being produced that– are more resistant to herbicides and pests and– provide nutrients that help address malnutrition.

▪ GM animals are being produced with improved nutritional or other qualities.


Figure 12.8B

12.9 Genetically modified organisms raise concerns about human and environmental health

▪ Scientists use safety measures to guard against production and release of new pathogens.

▪ Concerns related to GM organisms include the potential– introduction of allergens into the food supply and– spread of genes to closely related organisms.

▪ Regulatory agencies are trying to address the– safety of GM products,– labeling of GM produced foods, and– safe use of biotechnology.


12.10 CONNECTION: Gene therapy may someday help treat a variety of diseases

▪ Gene therapy aims to treat a disease by supplying a functional allele.

▪ One possible procedure is the following: 1. Clone the functional allele and insert it in a retroviral

vector.

2. Use the virus to deliver the gene to an affected cell type from the patient, such as a bone marrow cell.

3. Viral DNA and the functional allele will insert into the patient’s chromosome.

4. Return the cells to the patient for growth and division.



▪ Gene therapy is an– alteration of an afflicted individual’s genes and– attempt to treat disease.

▪ Gene therapy may be best used to treat disorders traceable to a single defective gene.


Figure 12.10

An RNA version ofa normal humangene is insertedinto a retrovirus.

RNA genome of virus

Retrovirus

Bone marrow cellsare infected withthe virus.

Viral DNA carrying thehuman gene inserts intothe cell’s chromosome.

Bone marrowcell from the patient

Bonemarrow

The engineeredcells are injectedinto the patient.

Cloned gene(normal allele) 1

2

3

4

Figure 12.10_1

An RNA version ofa normal humangene is insertedinto a retrovirus.

RNA genome of virus

Retrovirus

Cloned gene(normal allele) 1

Figure 12.10_2

Bone marrow cellsare infected withthe virus.

Viral DNA carrying thehuman gene inserts intothe cell’s chromosome.

Bone marrowcell from the patient

Bonemarrow

The engineeredcells are injectedinto the patient.

2

3

4


▪ The first successful human gene therapy trial in 2000– tried to treat ten children with SCID (severe combined

immune deficiency),– helped nine of these patients, but– caused leukemia in three of the patients, and– resulted in one death.



▪ The use of gene therapy raises many questions.– How can we build in gene control mechanisms that

make appropriate amounts of the product at the right time and place?

– How can gene insertion be performed without harming other cell functions?

– Will gene therapy lead to efforts to control the genetic makeup of human populations?

– Should we try to eliminate genetic defects in our children and descendants when genetic variety is a necessary ingredient for the survival of a species?


DNA PROFILING


12.11 The analysis of genetic markers can produce a DNA profile

▪ DNA profiling is the analysis of DNA fragments to determine whether they come from the same individual. DNA profiling– compares genetic markers from noncoding regions that

show variation between individuals and

– involves amplifying (copying) of markers for analysis.


Figure 12.11

DNA isisolated.

1

2

3

The DNA ofselectedmarkers isamplified.

The amplifiedDNA iscompared.

Crime scene Suspect 1 Suspect 2

12.12 The PCR method is used to amplify DNA sequences

▪ Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule.

▪ PCR relies upon a pair of primers that are– short, – chemically synthesized, single-stranded DNA

molecules, and– complementary to sequences at each end of the target

sequence.▪ PCR

– is a three-step cycle that– doubles the amount of DNA in each turn of the cycle.


Figure 12.12

Cycle 1yields two molecules

Cycle 2yields four molecules

DNApolymeraseaddsnucleotides.

Primers bondwith endsof targetsequences.

HeatseparatesDNAstrands.

GenomicDNA

Targetsequenc

e

Primer New DNA

Cycle 3yields eight molecules

3′

5′

5′

3′

3′5′ 5′

5′

5′5′

5′3′ 3′

3′ 3′

5′3′

5′

3′3′5′

5′

321

Figure 12.12_1

Cycle 1yields two molecules

DNApolymeraseaddsnucleotides.

Primers bondwith endsof targetsequences.

HeatseparatesDNAstrands.

GenomicDNA

Targetsequence

Primer New DNA

5′

3′

3′

5′

5′

3′ 3′ 5′

5′

5′ 3′ 5′

5′ 3′

5′3′ 3′

5′

5′3′

5′ 3′321

Figure 12.12_2

Cycle 2yields four molecules

Cycle 3yields eight molecules

▪ The advantages of PCR include– the ability to amplify DNA from a small sample,

– obtaining results rapidly, and

– a reaction that is highly sensitive, copying only the target sequence.

12.12 The PCR method is used to amplify DNA sequences


12.13 Gel electrophoresis sorts DNA molecules by size

▪ Gel electrophoresis can be used to separate DNA molecules based on size as follows:1. A DNA sample is placed at one end of a porous gel.2. Current is applied and DNA molecules move from the

negative electrode toward the positive electrode.3. Shorter DNA fragments move through the gel matrix

more quickly and travel farther through the gel. 4. DNA fragments appear as bands, visualized through

staining or detecting radioactivity or fluorescence.5. Each band is a collection of DNA molecules of the

same length.


Figure 12.13

A mixture of DNAfragments ofdifferent sizes

Powersource

Gel

Completedgel

Longer(slower)molecules

Shorter(faster)molecules

Figure 12.13_1

A mixture of DNAfragments ofdifferent sizes

Powersource

Gel

Completedgel

Longer(slower)molecules

Shorter(faster)molecules

Figure 12.13_2

12.14 STR analysis is commonly used for DNA profiling

▪ Repetitive DNA consists of nucleotide sequences that are present in multiple copies in the genome.

▪ Short tandem repeats (STRs) are short nucleotide sequences that are repeated in tandem,– composed of different numbers of repeating units in

individuals and– used in DNA profiling.

▪ STR analysis– compares the lengths of STR sequences at specific sites

in the genome and– typically analyzes 13 different STR sites.


Figure 12.14A

Crime sceneDNA

Suspect’sDNA

STR site 1 STR site 2

The number of shorttandem repeats match

The number of short tandemrepeats do not match

Figure 12.14B

CrimesceneDNA

Suspect’sDNA

Longer STR fragments

Shorter STR fragments

12.15 CONNECTION: DNA profiling has provided evidence in many forensic investigations

▪ DNA profiling is used to– determine guilt or innocence in a crime,

– settle questions of paternity,

– identify victims of accidents, and

– probe the origin of nonhuman materials.


Figure 12.15B

Cheddar Man and one of his modern-day descendants

12.16 RFLPs can be used to detect differences in DNA sequences

▪ A single nucleotide polymorphism (SNP) is a variation at a single base pair within a genome.

▪ Restriction fragment length polymorphism (RFLP) is a change in the length of restriction fragments due to a SNP that alters a restriction site.

▪ RFLP analysis involves– producing DNA fragments by restriction enzymes and

– sorting these fragments by gel electrophoresis.


Figure 12.16Restrictionenzymes

addedDNA sample

1DNA sample

2

CutCut

Cut

w

x

y y

z

Sample1

Sample2

z

x

wy y

Longerfragments

Shorterfragments

Figure 12.16_1

Restrictionenzymes

addedDNA sample 1 DNA sample

2w

x

y y

z

Cut

Cut Cut

Figure 12.16_2

Sample1

z

x

wy y

Longerfragments

Shorterfragments

Sample2

GENOMICS


12.17 Genomics is the scientific study of whole genomes

▪ Genomics is the study of an organism’s complete set of genes and their interactions.

– Initial studies focused on prokaryotic genomes.

– Many eukaryotic genomes have since been investigated.


Table 12.17

12.17 Genomics is the scientific study of whole genomes

▪ Genomics allows another way to examine evolutionary relationships.– Genomic studies showed a 96% similarity in DNA

sequences between chimpanzees and humans.

– Functions of human disease-causing genes have been determined by comparing human genes to similar genes in yeast.


12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes

▪ The goals of the Human Genome Project (HGP) included– determining the nucleotide sequence of all DNA in the

human genome and

– identifying the location and sequence of every human gene.


12.18 CONNECTION: The Human Genome Project revealed that most of the human genome does not consist of genes

▪ Results of the Human Genome Project indicate that– humans have about 20,000 genes in 3.2 billion

nucleotide pairs,– only 1.5% of the DNA codes for proteins, tRNAs, or

rRNAs, and– the remaining 98.5% of the DNA is noncoding DNA

including– telomeres, stretches of noncoding DNA at the ends of

chromosomes, and– transposable elements, DNA segments that can move or be

copied from one location to another within or between chromosomes.


Figure 12.18Exons (regions of genes coding for protein

or giving rise to rRNA or tRNA) (1.5%)

RepetitiveDNA thatincludestransposableelementsand relatedsequences(44%)

Introns andregulatorysequences(24%)

UniquenoncodingDNA (15%)

RepetitiveDNAunrelated totransposableelements(15%)

12.19 The whole-genome shotgun method of sequencing a genome can provide a wealth of data quickly

▪ The Human Genome Project proceeded through three stages that provided progressively more detailed views of the human genome.1. A low-resolution linkage map was developed using

RFLP analysis of 5,000 genetic markers.

2. A physical map was constructed from nucleotide distances between the linkage-map markers.

3. DNA sequences for the mapped fragments were determined.



▪ The whole-genome shotgun method– was proposed in 1992 by molecular biologist J. Craig

Venter, who

– used restriction enzymes to produce fragments that were cloned and sequenced in just one stage and

– ran high-performance computer analyses to assemble the sequence by aligning overlapping regions.



▪ Today, this whole-genome shotgun approach is the method of choice for genomic researchers because it is– relatively fast and– inexpensive.

▪ However, limitations of the whole-genome shotgun method suggest that a hybrid approach using genome shotgunning and physical maps may prove to be the most useful.


Figure 12.19

ChromosomeChop up each chromosomewith restriction enzymes

Sequence the fragments

DNA fragments

Align the fragments

Reassemble the fullsequence

12.20 Proteomics is the scientific study of the full set of proteins encoded by a genome

▪ Proteomics– is the study of the full protein sets encoded by

genomes and

– investigates protein functions and interactions.

▪ The human proteome includes about 100,000 proteins.

▪ Genomics and proteomics are helping biologists study life from an increasingly holistic approach.


12.21 EVOLUTION CONNECTION: Genomes hold clues to human evolution

▪ Human and chimp genomes differ by– 1.2% in single-base substitutions and– 2.7% in insertions and deletions of larger DNA

sequences.

▪ Genes showing rapid evolution in humans include– genes for defense against malaria and tuberculosis,– a gene regulating brain size, and– the FOXP2 gene involved with speech and vocalization.


12.21 EVOLUTION CONNECTION: Genomes hold clues to human evolution

▪ Neanderthals– were close human relatives,– were a separate species,– also had the FOXP2 gene,– may have had pale skin and red hair, and– were lactose intolerant.


Figure 12.21

Reconstruction of a Neanderthal female, based on a 36,000 year old skull

1. Explain how plasmids are used in gene cloning.

2. Explain how restriction enzymes are used to “cut and paste” DNA into plasmids.

3. Explain how plasmids, phages, and BACs are used to construct genomic libraries.

4. Explain how a cDNA library is constructed and how it is different from genomic libraries constructed using plasmids or phages.

5. Explain how a nucleic acid probe can be used to identify a specific gene.

You should now be able to


6. Explain how different organisms are used to mass-produce proteins of human interest.

7. Explain how DNA technology has helped to produce insulin, growth hormone, and vaccines.

8. Explain how genetically modified (GM) organisms are transforming agriculture.

9. Describe the risks posed by the creation and culturing of GM organisms and the safeguards that have been developed to minimize these risks.



10. Describe the benefits and risks of gene therapy in humans. Discuss the ethical issues that these techniques present.

11. Describe the basic steps of DNA profiling.

12. Explain how PCR is used to amplify DNA sequences.

13. Explain how gel electrophoresis is used to sort DNA and proteins.

14. Explain how short tandem repeats are used in DNA profiling.



15. Describe the diverse applications of DNA profiling.

16. Explain how restriction fragment analysis is used to detect differences in DNA sequences.

17. Explain why it is important to sequence the genomes of humans and other organisms.

18. Describe the structure and possible functions of the noncoding sections of the human genome.

19. Explain how the human genome was mapped.



21. Compare the fields of genomics and proteomics.

22. Describe the significance of genomics to the study of evolutionary relationships and our understanding of the special characteristics of humans.



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