PowerLecture:PowerLecture:Chapter 16Chapter 16

Studying and Manipulating Studying and Manipulating GenomesGenomes

Fig. 16-1a, p.242

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

Ordinary daffodil

124 million children around the world have 124 million children around the world have vitamin A deficienciesvitamin A deficiencies

Golden rice Golden rice ––Rice plants engineered with genes from daffodils Rice plants engineered with genes from daffodils

allowing it to produce beta-carotine in its seeds allowing it to produce beta-carotine in its seeds (rice) (rice)

––Beta carotine is the precursor to Vitamin ABeta carotine is the precursor to Vitamin A

Rice is the main food for 3 billion peopleRice is the main food for 3 billion people

Impacts, Issues: Impacts, Issues: Golden Rice, or Golden Rice, or Frankenfood?Frankenfood?

Fig. 16-1b, p.242

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

Regular rice

1st generation2nd generation

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 practicesthan current agricultural practices

Impacts, Issues: Impacts, Issues: Golden Rice, or Golden Rice, or Frankenfood?Frankenfood?

p.243

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

Today's cornAncestral corn

Discovery of Restriction Discovery of Restriction EnzymesEnzymes

Hamilton Smith was studying how Hamilton Smith was studying how Haemophilus influenzaeHaemophilus influenzae defend defend themselves from bacteriophage attackthemselves from bacteriophage attack

Discovered bacteria have an enzyme that Discovered bacteria have an enzyme that chops up viral DNA chops up viral DNA

Specificity of CutsSpecificity of Cuts

Restriction enzymes cut DNA at a Restriction enzymes cut DNA at a specific sequencespecific sequence

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 occurssequence occurs

3’

5’ 3’

5’GC T T A A

A A T T C

G

another DNA fragment

5’

3’

G

one DNA fragment

3’

5’C T T A A

A A T T C

nick

nick

G A A T T C

GC T T A A3’

5’ 3’

5’

DNA ligase action

GC T T A A3’

5’ 3’

5’

A A T T CG

Fig. 16-2, p.244

TermsTerms

DNA ligase – seals cuts in DNADNA ligase – seals cuts in DNA Recombinant DNA – any molecule Recombinant DNA – any molecule

consisting of base sequences from 2 or consisting of base sequences from 2 or more organisms of the same or different more organisms of the same or different speciesspecies

Cloning vector – a plasmid that has Cloning vector – a plasmid that has accepted foreign DNA and can be slipped accepted foreign DNA and can be slipped into hostinto host

Using PlasmidsUsing 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

Fig. 16-3a, p.244

PlasmidsPlasmids

a A restriction enzymecuts a specific base sequence everywhere it occurs in DNA.

c The same enzyme cuts the same sequnece in plasmid DNA.

d The plasmid DNA also has sticky ends

e The DNA fragments and the plasmid DNA are mixed with DNA ligase.

f The result? A collection of recombinant plasmids that incorporate foreign DNA fragments.

g Host cells that can divide rapidly take up the recombinant plasmids.

b The DNA fragmentshave sticky ends.

Fig. 16-4, p.245

Using Plasmids

Making cDNAMaking cDNA

Fig. 16-5, p.245

•Use reverse transcriptase onto one strand of complementary DNA (cDNA)•DNA polymerase strips RNA bases•Copies cDNA into a second strand

Gene LibrariesGene Libraries

Bacteria that contain different Bacteria that contain different

cloned DNA fragmentscloned DNA fragments

Genomic libraryGenomic library

cDNA librarycDNA library

Use of a ProbeUse of a Probe Colonies on plate

Cells adhere to filter

Cells are lysed;DNA sticks to filter

Probe is added

Location where probe binds forms dark spot on film, indicates colony with gene

You want to find You want to find

which bacteria in a which bacteria in a

library contain a library contain a

specific genespecific gene

Need a probe for that Need a probe for that

genegene

• A radioisotope-A radioisotope-

labeled piece of labeled piece of

DNADNA

• Will base-pair with Will base-pair with

gene of interestgene of interest

Making a ProbeMaking a Probe

Make a primer if the sequence is known.Make a primer if the sequence is known. If not usually DNA from closely related speciesIf not usually DNA from closely related species

Amplifying DNAAmplifying DNA

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

Polymerase chain reaction (PCR) Polymerase chain reaction (PCR)

Polymerase Chain ReactionPolymerase Chain Reaction

Sequence to be copied is heatedSequence to be copied is heated Primers are added and bind to ends of Primers are added and bind to ends of

single strandssingle strands DNA polymerase uses free nucleotides DNA polymerase uses free nucleotides

to create complementary strandsto create complementary strands Doubles number of copies of DNADoubles number of copies of DNA Animation

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

Recording Recording the 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

Fig. 16-8b, p.248

Recording the Sequence

http://www.dnalc.org/ddnalc/resources/cycseq.html

Genome SequencingGenome 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

Fig. 16-10a, p.250

Genome SequencingGenome Sequencing

Nucleotides for SequencingNucleotides for Sequencing

Standard nucleotides (A, T, C, G)Standard nucleotides (A, T, C, G) Modified versions of these nucleotides Modified versions of these nucleotides

Labeled so they fluoresceLabeled so they fluoresce Structurally different so that they stop DNA Structurally different so that they stop DNA

synthesis when they are added to a strandsynthesis when they are added to a strand

Reaction MixtureReaction Mixture

Copies of DNA to be sequencedCopies of DNA to be sequenced PrimerPrimer DNA polymeraseDNA polymerase Standard nucleotidesStandard nucleotides Modified nucleotidesModified nucleotides

DNA FingerprintsDNA Fingerprints

Unique array of DNA fragmentsUnique array of DNA fragments

Inherited from parents in Mendelian Inherited from parents in Mendelian

fashionfashion

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

Short regions of DNA that differ Short regions of DNA that differ substantially among peoplesubstantially among people

Many sites in genome where tandem Many sites in genome where tandem repeats occurrepeats 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 sizevary in size

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 http://www.dnalc.org/ddnalc/resources/elec

trophoresis.html

Fig. 16-9b, p.249

Gel Gel ElectrophoresisElectrophoresis

Analyzing DNA Fingerprints Analyzing DNA Fingerprints

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

of a radioactive probeof a 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 bodiesIdentify bodies

Determine paternityDetermine paternity

GenomicsGenomics

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

Reactions ProceedReactions Proceed

Nucleotides are assembled to create Nucleotides are assembled to create complementary strandscomplementary strands

When a modified nucleotide is included, When a modified nucleotide is included, synthesis stopssynthesis stops

Result is millions of tagged copies of Result is millions of tagged copies of varying lengthvarying length

DNA ChipsDNA Chips

Microarrays of Microarrays of thousands of gene thousands of gene sequences sequences representing a large representing a large subset of an entire subset of an entire genome (p251)genome (p251)

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

Genetic EngineeringGenetic Engineering

Genes are isolated, modified, and inserted Genes are isolated, modified, and inserted into an organisminto an organism

Made possible by recombinant technologyMade possible by recombinant technology

Cut DNA up and recombine piecesCut DNA up and recombine pieces

Amplify modified pieces Amplify modified pieces

Engineered ProteinsEngineered Proteins

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

valuable proteinsvaluable proteins

Insulin, interferon, blood-clotting factorsInsulin, interferon, blood-clotting factors

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 can be engineered to break down Some can be engineered to break down

pollutants or to take up larger amounts pollutants or to take up larger amounts

of harmful materialsof harmful materials

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

p.252

Engineered PlantsEngineered Plants

Cotton plants that display resistance to Cotton plants that display resistance to herbicideherbicide

Aspen plants that produce less lignin and Aspen plants that produce less lignin and more cellulosemore cellulose

Tobacco plants that produce human Tobacco plants that produce human proteinsproteins

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

Fig. 16-12a, p.253

Engineered Plants

Fig. 16-12b, p.253

Engineered Plants

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 plasmid

Genetic ChangesGenetic Changes

Humans have been changing the Humans have been changing the

genetics of other species for thousands genetics of other species for thousands

of yearsof years Artificial selection of plants and animalsArtificial selection of plants and animals

Natural processes also at workNatural processes also at work Mutation, crossing overMutation, crossing over

First Engineered MammalsFirst 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

Transgenic MiceTransgenic Mice

Fig. 16-15, p.254

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

Fig. 16-14a, p.254

Genetically Modified Animals

Fig. 16-14b, p.254

Genetically Modified Animals

Fig. 16-14c, p.254

Genetically Modified Animals

XenotransplantationXenotransplantation

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

SafetySafety

SuperpathogensSuperpathogens DNA from pathogenic or toxic organisms DNA from pathogenic or toxic organisms

used in recombination experimentsused in recombination experiments Hok genesHok genes NIH guidelines for DNA researchNIH guidelines for DNA research

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 2001schedule in early 2001

Results of Gene TherapyResults of Gene Therapy

Modified cells alive in woman’s liverModified cells alive in woman’s liver

Blood levels of LDLs down 20 percentBlood levels of LDLs down 20 percent

No evidence of atherosclerosis No evidence of atherosclerosis

Cholesterol levels remain highCholesterol levels remain high

Remains to be seen whether procedure will Remains to be seen whether procedure will

prolong her lifeprolong her life

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 usually used to insert genes Viruses usually used to insert genes into cultured human cells but procedure into cultured human cells but procedure has problemshas problems

Very difficult to get modified genes to Very difficult to get modified genes to work where they shouldwork 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?