chapter 16 studying and manipulating genomes. objectives 1. know how dna can be cleaved, spliced,...

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Chapter 16 Chapter 16 Studying and Manipulating Studying and Manipulating Genomes Genomes

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Chapter 16Chapter 16

Studying and Manipulating Studying and Manipulating GenomesGenomes

ObjectivesObjectives

1. Know how DNA can be cleaved, spliced, cloned, and sequenced.

2. Understand what plasmids are and how they may be used to insert new genes into recombinant DNA molecules.

3. Explain the types of genetic experiments that nature has been performing for billions of years.

Objectives – cont.Objectives – cont.

4. Understand how one organism can produce the products of another.

5. Be aware of several limits and possibilities for future research in genetic engineering.

6. Explain how a new field of research called genomics has come about from the Human Genome Project.

124 million children around the world have 124 million children around the world have vitamin A deficiencies, they do not grow or vitamin A deficiencies, they do not grow or develop normally, and may become blind.develop normally, and may become blind.

Three genes (beta carotene –yellow pigment) Three genes (beta carotene –yellow pigment) from daffodils have been transferred into rice from daffodils have been transferred into rice plants.plants.

Rice plants engineered with genes produce Rice plants engineered with genes produce beta-carotene in its seeds ( Golden rice) beta-carotene in its seeds ( Golden rice)

Beta carotene is the precursor to Vitamin ABeta carotene is the precursor to Vitamin A Rice is the main food for 3 billion peopleRice is the main food for 3 billion people

16.016.0Golden Rice, or Frankenfood?Golden Rice, or Frankenfood?

Humans have been experimenting with Humans have been experimenting with new breeds of organisms for thousands of new breeds of organisms for thousands of years.years.

Many crops plants have been modified, Many crops plants have been modified, including corn, beets, potatoes, and cottonincluding corn, beets, potatoes, and cotton

Potentially less harmful to the environment Potentially less harmful to the environment than current agricultural practices – less than current agricultural practices – less pesticide is needed.pesticide is needed.

Golden Rice, or Frankenfood?Golden Rice, or Frankenfood?

16.1 A Molecular Toolkit16.1 A Molecular Toolkit

In 1970, Hamilton Smith was studying how In 1970, Hamilton Smith was studying how Haemophilus influenzaeHaemophilus influenzae (bacteria) defend (bacteria) defend themselves from bacteriophage attackthemselves from bacteriophage attack

He discovered bacteria have an enzyme He discovered bacteria have an enzyme that chops up viral DNA (foreign that chops up viral DNA (foreign molecules) before it can get inserted into molecules) before it can get inserted into the bacterial chromosome.the bacterial chromosome.

Restriction EnzymesRestriction Enzymes

Restriction enzymes (scissors) cut DNA Restriction enzymes (scissors) cut DNA at a specific base sequence (4 to 8 at a specific base sequence (4 to 8 base pairs in length).base pairs in length).

Number of cuts made in DNA will Number of cuts made in DNA will depend on number of times the “target” depend on number of times the “target” sequence occurs, in the 5’ sequence occurs, in the 5’ 3’ 3’ direction on both strands of DNA.direction on both strands of DNA.

Restriction EnzymesRestriction Enzymes

Many times the “sticky ends” that results Many times the “sticky ends” that results from the cut can be used to pair up with from the cut can be used to pair up with another DNA fragment cut by the same another DNA fragment cut by the same enzyme.enzyme.

DNA fragments produced by restriction DNA fragments produced by restriction enzymes are treated with DNA ligase to enzymes are treated with DNA ligase to splice the DNA fragments together to form splice the DNA fragments together to form a recombinant DNA molecule.a recombinant DNA molecule.

Fig. 16-2, p.244

Stepped ArtGC T T A A

A A T T CG

3’

5’ 3’

5’

C T T A A

A A T T CG

cut fragments

G

DNA ligase action

nick

nick

GC T T A A

A A T T CG

3’

5’ 3’

5’

another DNA fragment

A A T T C

3’

5’ 3’

5’G5’

one DNA fragment

3’

3’

5’ G

C T T A A

3’

5’

enzyme recognition site

GC T T A A

A A T T CG 3’

5’

Cloning VectorsCloning Vectors

Plasmids are circular DNA molecules in Plasmids are circular DNA molecules in bacteria that carry only a few genes and bacteria that carry only a few genes and can replicate independently of the single can replicate independently of the single “main” chromosome.“main” chromosome.

Modified plasmids that are capable of Modified plasmids that are capable of accepting, replicating, and delivering DNA accepting, replicating, and delivering DNA to another host cell are called cloning to another host cell are called cloning vectors.vectors.

Cloning Vectors -Using PlasmidsCloning Vectors -Using Plasmids

Plasmid is small circle of Plasmid is small circle of

bacterial DNAbacterial DNA

Foreign DNA can be inserted Foreign DNA can be inserted

into plasmidinto plasmid

Forms recombinant plasmidsForms recombinant plasmids

Plasmid is a cloning vectorPlasmid is a cloning vector

Can deliver DNA into another cellCan deliver DNA into another cell

cDNA cloningcDNA cloning

Researches use DNA made from “mature” Researches use DNA made from “mature” mRNA transcripts ( i.e. introns have been mRNA transcripts ( i.e. introns have been removed from original DNA)removed from original DNA)

Restriction enzymes will not cut single-Restriction enzymes will not cut single-stranded molecules.stranded molecules.

Reverse transcriptase catalyzes the Reverse transcriptase catalyzes the assembly of a single DNA strand on an assembly of a single DNA strand on an mRNA template, forming a hybrid.mRNA template, forming a hybrid.

cDNA (cont.)cDNA (cont.)

DNA polymerase replaces the mRNA with DNA polymerase replaces the mRNA with another DNA strand.another DNA strand.

The result is a double stranded DNA, that The result is a double stranded DNA, that may be inserted into a plasmid for may be inserted into a plasmid for amplification or cloning.amplification or cloning.

cDNA CloningcDNA Cloning

Fig. 16-5, p.245

16.2 Gene Libraries16.2 Gene Libraries

A gene library is a collection of host A gene library is a collection of host

cells (bacteria) that house different cells (bacteria) that house different

cloned fragments of DNA.cloned fragments of DNA.

A genomic library are the cloned A genomic library are the cloned

fragments of an entire genome.fragments of an entire genome.

A cDNA library is derived from mRNA.A cDNA library is derived from mRNA.

Using a Probe to Find a GeneUsing a Probe to Find a Gene

You want to find which bacteria in a You want to find which bacteria in a

library contain a specific genelibrary contain a specific gene

Need a probe for that geneNeed a probe for that gene

A radioisotope-labeled piece of DNA A radioisotope-labeled piece of DNA

that will base-pair with gene of that will base-pair with gene of

interestinterest

a Bacterial colonies, each derived from a single cell, grow on a culture plate. Each colony is about 1 millimeter across.

b A nitrocellulose or nylon filter is placed on the plate. Somecells of each colony adhere to it. The filter mirrors how the colonies are distributed on the culture plate.

c The filter is lifted off and put into a solution. Cells stuck to it rupture; the cellular DNA sticks to the filter.

d The DNA is denatured to single strands at each site. A radioactively labeled probe is added to the filter. The probe binds to DNA with a complementary base sequence.

e The probe’s location is identified by exposing the filter to x-ray film. The image that forms on the film reveals the colony that has the gene of interest.

Fig. 16-6, p.246

Use of a ProbeUse of a Probe

Amplifying DNAAmplifying DNA

Fragments can be inserted into Fragments can be inserted into fast-growing microorganisms fast-growing microorganisms

Polymerase chain reaction (PCR) is Polymerase chain reaction (PCR) is a method of rapidly and a method of rapidly and exponentially amplifying the number exponentially amplifying the number of particular DNA fragments, used to of particular DNA fragments, used to make millions of copies of cDNA. make millions of copies of cDNA.

Polymerase Chain ReactionPolymerase Chain Reaction

Sequence to be copied is heatedSequence to be copied is heated Primers (short nucleotide sequences) Primers (short nucleotide sequences)

are added and bind to ends of single are added and bind to ends of single strands. They are start tags.strands. They are start tags.

DNA polymerase (heat resistant) uses DNA polymerase (heat resistant) uses free nucleotides to create free nucleotides to create complementary strandscomplementary strands

Doubles number of copies of DNADoubles number of copies of DNA

Polymerase Polymerase Chain ReactionChain Reaction

Double-stranded DNA to copy

DNA heated to 90°– 94°C

Primers added to base-pair with ends

Mixture cooled; base-pairing of primers and ends of DNA strands

DNA polymerasesassemble new DNA strands

Fig. 16-6, p. 256

Stepped Art

Polymerase Polymerase Chain ReactionChain Reaction

Stepped Art

Mixture heated again; makes all DNA fragments unwind

Mixture cooled; base-pairing between primers and ends of single DNA strands

DNA polymerase action again doubles number of identical DNA fragments

Fig. 16-6, p. 256

16.3 Automated DNA 16.3 Automated DNA sequencingsequencing

This techniques used DNA polymerase to This techniques used DNA polymerase to partially replicate a DNA template.partially replicate a DNA template.

Laboratories use automated DNA Laboratories use automated DNA sequencing to determine the unknown sequencing to determine the unknown sequence of bases in any DNA sample sequence of bases in any DNA sample (cloned or PCR-amplified DNA) in just a (cloned or PCR-amplified DNA) in just a few hours.few hours.

Automated DNA sequencingAutomated DNA sequencing

The machine builds DNA molecules but uses The machine builds DNA molecules but uses eight kinds of bases: four normal (A, T, C, G) eight kinds of bases: four normal (A, T, C, G) and four that are modified to fluoresce in and four that are modified to fluoresce in different colors in laser light, Structurally different colors in laser light, Structurally different so that they stop DNA synthesis when different so that they stop DNA synthesis when they are added to a strand.they are added to a strand.

Researchers mix these eight bases with a single Researchers mix these eight bases with a single stranded DNA template, a primer, and DNA stranded DNA template, a primer, and DNA polymerasepolymerase..

Automated DNA sequencingAutomated DNA sequencing

The automated DNA sequencer separates The automated DNA sequencer separates the set of fragments by gel the set of fragments by gel electrophoresis.electrophoresis.

The “tag” base at the end of each The “tag” base at the end of each fragment in the set is identified by the fragment in the set is identified by the laser beam.laser beam.

The computer program assembles the The computer program assembles the information to reveal the entire DNA information to reveal the entire DNA sequence.sequence.

Recording the Recording the Sequence Sequence

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

T C C A T G G A

T C C A T G G

T C C A T G

T C C A T

T C C A

T C C

T C

T

electrophoresisgel

one of the many fragments of DNA migratingthrough the gel

one of the DNA fragmentspassing through a laser beam after moving through the gel

T C C A T G G A C C A

•DNA is placed on gel

•Fragments move off

gel in size order; pass

through laser beam

•Color each fragment

fluoresces is recorded

on printout

16.4 DNA Fingerprints16.4 DNA Fingerprints

They are unique array of DNA fragmentsThey are unique array of DNA fragments

Inherited from parents in Mendelian Inherited from parents in Mendelian

fashionfashion

More than 99% of DNA is the same in all More than 99% of DNA is the same in all

humans – only 1% is differenthumans – only 1% is different

Even full siblings can be distinguished Even full siblings can be distinguished

from one another by this technique from one another by this technique

Tandem RepeatsTandem Repeats

The technique focuses on tandem The technique focuses on tandem repeats – copies of the same short repeats – copies of the same short regions of DNA that differ substantially regions of DNA that differ substantially among peopleamong people

There are many sites in genome where There are many sites in genome where tandem repeats occurtandem repeats occur

Each person carries a unique Each person carries a unique combination of repeat numberscombination of repeat numbers

RFLPsRFLPs

Restriction fragment length polymorphismsRestriction fragment length polymorphisms DNA from areas with tandem repeats is cut DNA from areas with tandem repeats is cut

with restriction enzymeswith restriction enzymes Because of the variation in the amount of Because of the variation in the amount of

repeated DNA, the restriction fragments repeated DNA, the restriction fragments vary in size (vary by individual)vary in size (vary by individual)

Variation is detected by gel electrophoresisVariation is detected by gel electrophoresis

Gel ElectrophoresisGel Electrophoresis

DNA is placed at one end of a gelDNA is placed at one end of a gel A current is applied to the gelA current is applied to the gel DNA molecules are negatively charged DNA molecules are negatively charged

and move toward positive end of geland move toward positive end of gel Smaller molecules move faster than larger Smaller molecules move faster than larger

onesones DNA may be amplified by PCR if the DNA may be amplified by PCR if the

sample is small.sample is small.

Fig. 16-9b, p.249

Gel Gel ElectrophoresisElectrophoresis

Analyzing DNA Fingerprints Analyzing DNA Fingerprints

DNA is stained or made visible by use of DNA is stained or made visible by use of

a radioactive probea radioactive probe

Pattern of bands is used to: Pattern of bands is used to:

Identify or rule out criminal suspectsIdentify or rule out criminal suspects

Identify bodies – 9/11 victimsIdentify bodies – 9/11 victims

Determine paternityDetermine paternity

16.5 Genome Sequencing16.5 Genome Sequencing

1995 - Sequence of bacterium 1995 - Sequence of bacterium Haemophilus influenzaeHaemophilus influenzae determined determined

Automated DNA sequencing now main Automated DNA sequencing now main methodmethod

Draft sequence of entire human genome Draft sequence of entire human genome determined in this waydetermined in this way

Human Genome Project began in 1988 Human Genome Project began in 1988

The Human Genome InitiativeThe Human Genome Initiative

Goal - Map the entire human genomeGoal - Map the entire human genome Initially thought by many to be a waste of Initially thought by many to be a waste of

resourcesresources Process accelerated when Craig Ventner Process accelerated when Craig Ventner

used bits of cDNAs as hooks to find genesused bits of cDNAs as hooks to find genes Sequencing was completed ahead of Sequencing was completed ahead of

schedule in early 2003schedule in early 2003

Genome sequencingGenome sequencing

About 99% of the coding regions of human About 99% of the coding regions of human DNA have been decipheredDNA have been deciphered

About 20,000 confirmed genesAbout 20,000 confirmed genes Protein encoding genes make up less than Protein encoding genes make up less than

2 percent of our genome.2 percent of our genome.

GenomicsGenomics

Genomics is a new research field.Genomics is a new research field.

Structural genomics: actual mapping and Structural genomics: actual mapping and sequencing of genomes of individuals sequencing of genomes of individuals

Comparative genomics: concerned with Comparative genomics: concerned with possible evolutionary relationships of possible evolutionary relationships of groups of organismsgroups of organisms

GenomicsGenomics

Genomics has potential for human gene Genomics has potential for human gene therapy – the transfer of one or more therapy – the transfer of one or more normal or modified genes into a person’s normal or modified genes into a person’s body cells to correct a genetic defect or body cells to correct a genetic defect or boost resistance to disease.boost resistance to disease.

Some gene therapies use viruses as Some gene therapies use viruses as vectors, deliver modified cells into a vectors, deliver modified cells into a patients tissue.patients tissue.

DNA ChipsDNA Chips

Researchers can pinpoint which genes are Researchers can pinpoint which genes are silent and which are being expressed with silent and which are being expressed with the use of DNA chips.the use of DNA chips.

DNA Chips are microarrays of thousands DNA Chips are microarrays of thousands of gene sequences representing a large of gene sequences representing a large subset of an entire genome subset of an entire genome

Stamped onto a glass plate the size of a Stamped onto a glass plate the size of a small business card (p251)small business card (p251)

DNA ChipsDNA Chips

DNA chips are being used to compare DNA chips are being used to compare different genes expression patterns different genes expression patterns between cells. Examples are yeasts between cells. Examples are yeasts grown in the presence or absence of grown in the presence or absence of oxygen. See fig. 16.11oxygen. See fig. 16.11

Green – genes active during fermentationGreen – genes active during fermentation Red – genes used in aerobic respirationRed – genes used in aerobic respiration Yellow – genes active in bothYellow – genes active in both

16.6 Genetic Engineering16.6 Genetic Engineering

Genetic Engineering is the deliberate Genetic Engineering is the deliberate modification of an individual’s genome.modification of an individual’s genome.

Genes from another species may be Genes from another species may be transferred to an individual.transferred to an individual.

The individual may have its own genes The individual may have its own genes isolated, modified and copied, and then isolated, modified and copied, and then receive copies of the modified genes.receive copies of the modified genes.

16.6 Genetic Engineering16.6 Genetic Engineering

Genetic engineering started with bacterial Genetic engineering started with bacterial species.species.

The kinds that take up plasmids are now The kinds that take up plasmids are now widely used in basic research, agriculture, widely used in basic research, agriculture, medicine and industry.medicine and industry.

Made possible by recombinant technologyMade possible by recombinant technology

Engineered ProteinsEngineered Proteins

Bacteria can be used to grow medically Bacteria can be used to grow medically

valuable proteinsvaluable proteins

Insulin, somatotropin (growth hormone), Insulin, somatotropin (growth hormone),

hemoglobin, interferon, blood-clotting hemoglobin, interferon, blood-clotting

factorsfactors

VaccinesVaccines

Cleaning Up the EnvironmentCleaning Up the Environment

Microorganisms normally break down Microorganisms normally break down

organic wastes and cycle materialsorganic wastes and cycle materials

Some bacteria can be engineered to Some bacteria can be engineered to

break down pollutants or to take up break down pollutants or to take up

larger amounts of harmful materials; oil, larger amounts of harmful materials; oil,

heavy metals, and radioactive wastes.heavy metals, and radioactive wastes.

16.7 Designer Plants16.7 Designer Plants

Pressured to produce more food at lower Pressured to produce more food at lower cost and with less damage to the cost and with less damage to the environment, farmers are turning to environment, farmers are turning to genetically engineered crop plants.genetically engineered crop plants.

This leads to a decrease in pesticide use This leads to a decrease in pesticide use that can harm humans, animals, and that can harm humans, animals, and beneficial insects.beneficial insects.

Engineered PlantsEngineered Plants

Cotton plants that display resistance to Cotton plants that display resistance to herbicide that will kill weeds.herbicide that will kill weeds.

Aspen plants that produce less lignin and Aspen plants that produce less lignin and more cellulose for paper makingmore cellulose for paper making

Tobacco plants that produce human Tobacco plants that produce human proteinsproteins

Mustard plant cells that produce Mustard plant cells that produce biodegradable plasticbiodegradable plastic

The Ti plasmidThe Ti plasmid

Researchers Researchers replace tumor-replace tumor-causing genes causing genes with beneficial with beneficial genes genes

Plasmid Plasmid transfers these transfers these genes to genes to cultured plant cultured plant cellscells

foreign genein plasmid

plant cell

Fig. 16-13, p.253

a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue).

b The bacterium infects a plant and transfers the Ti plasmid into it.

c The plant cell divides.

d Transgenic plants.

e Young plants with a fluorescent gene product.

The Ti plasmidThe Ti plasmid

16.8 First Engineered Mammals16.8 First Engineered Mammals

Experimenters used mice with hormone Experimenters used mice with hormone deficiency that leads to dwarfism deficiency that leads to dwarfism

Fertilized mouse eggs were injected Fertilized mouse eggs were injected with gene for rat growth hormone with gene for rat growth hormone

Gene was integrated into mouse DNAGene was integrated into mouse DNA Engineered mice were 1-1/2 times Engineered mice were 1-1/2 times

larger than unmodified littermateslarger than unmodified littermates

Cloning DollyCloning Dolly

1997 - A sheep cloned from an adult cell1997 - A sheep cloned from an adult cell

Nucleus from mammary gland cell was Nucleus from mammary gland cell was

inserted into enucleated egg inserted into enucleated egg

Embryo implanted into surrogate mother Embryo implanted into surrogate mother

Sheep is genetic replica of animal from Sheep is genetic replica of animal from

which mammary cell was takenwhich mammary cell was taken

Designer CattleDesigner Cattle

Genetically identical cattle embryos can be Genetically identical cattle embryos can be grown in culturegrown in culture

Embryos can be genetically modifiedEmbryos can be genetically modified create resistance to mad cow diseasecreate resistance to mad cow disease engineer cattle to produce human serum engineer cattle to produce human serum

albumin for medical usealbumin for medical use

Transgenic AnimalsTransgenic Animals

Transgenic Animals are used routinely for Transgenic Animals are used routinely for medical research.medical research.

They can be the source of medically They can be the source of medically valued proteins.valued proteins.

XenotransplantationXenotransplantation

This is the transfer of an organ from one This is the transfer of an organ from one species to another.species to another.

Researchers knockout the Ggta1genes in Researchers knockout the Ggta1genes in transgenic pigletstransgenic piglets

Ggta1 gene produces proteins that human Ggta1 gene produces proteins that human antibodies recognizeantibodies recognize

Pig’s organs are less prone to rejection by Pig’s organs are less prone to rejection by a humana human

16.9 Safety16.9 Safety

SuperpathogensSuperpathogens

DNA from pathogenic or toxic organisms DNA from pathogenic or toxic organisms used in recombination experimentsused in recombination experiments

NIH guidelines for DNA researchNIH guidelines for DNA research

Can Genetically Engineered Can Genetically Engineered Bacteria “Escape”?Bacteria “Escape”?

Genetically engineered bacteria are Genetically engineered bacteria are designed so that they cannot survive designed so that they cannot survive outside laboutside lab

Genes are included that will be turned Genes are included that will be turned on in outside environment, triggering on in outside environment, triggering deathdeath

16.10 Modified Human?16.10 Modified Human?

The goal of human gene therapy is to The goal of human gene therapy is to transfer normal or modified genes into transfer normal or modified genes into body cells to correct genetic defects.body cells to correct genetic defects.

There are risks associated with these There are risks associated with these procedures, as noted with “Bubble” procedures, as noted with “Bubble” children suffering from a severe immune children suffering from a severe immune deficiency.deficiency.

Eugenic engineering – selecting the most Eugenic engineering – selecting the most desired human traits.desired human traits.

Using Human GenesUsing Human Genes

Even with gene in hand it is difficult to Even with gene in hand it is difficult to manipulate it to advantagemanipulate it to advantage

Viruses are usually used to insert genes Viruses are usually used to insert genes into cultured human cells, but the into cultured human cells, but the procedure has problemsprocedure has problems

It is very difficult to get modified genes It is very difficult to get modified genes to work where they shouldto work where they should

Ethical IssuesEthical Issues

Who decides what should be Who decides what should be

“corrected” through genetic “corrected” through genetic

engineering?engineering?

Should animals be modified to provide Should animals be modified to provide

organs for human transplants?organs for human transplants?

Should humans be cloned?Should humans be cloned?