Recombinant Recombinant DNA and Genetic DNA and Genetic

EngineeringEngineeringChapter 16Chapter 16

Familial Familial HypercholesterolemiaHypercholesterolemia

Gene encodes protein that serves as Gene encodes protein that serves as

cell’s LDL receptorcell’s LDL receptor

Two normal alleles for the gene keep Two normal alleles for the gene keep

blood level of LDLs lowblood level of LDLs low

Two mutated alleles lead to Two mutated alleles lead to

abnormally high cholesterol levels & abnormally high cholesterol levels &

heart diseaseheart disease

Example of Gene Therapy Example of Gene Therapy

Woman with familial hypercholesterolemiaWoman with familial hypercholesterolemia

Part of her liver was removed Part of her liver was removed

Virus used to insert normal gene for LDL Virus used to insert normal gene for LDL

receptor into cultured liver cellsreceptor into cultured liver cells

Modified liver cells placed back in patientModified liver cells placed back in patient

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 Remains to be seen whether

procedure will prolong her lifeprocedure will prolong her life

Genetic ChangesGenetic Changes

Humans have been changing the Humans have been changing the

genetics of other species for genetics of other species for

thousands of yearsthousands of years Artificial selection of plants and Artificial selection of plants and

animalsanimals

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

Genetic EngineeringGenetic Engineering

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

Made possible by recombinant Made possible by recombinant technologytechnology

Cut DNA up and recombine piecesCut DNA up and recombine pieces

Amplify modified pieces Amplify modified pieces

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 themselves from bacteriophage attackattack

Discovered bacteria have an enzyme Discovered bacteria have an enzyme that chops up viral DNA that 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 depend on number of times the “target” sequence occurs“target” sequence occurs

Making Recombinant Making Recombinant DNADNA

5’

3’

G

C T T A A

A A T T C

G

G A A T T C

C T T A A G3’

5’

one DNA fragment another DNA fragment

3’

5’

In-text In-text figurefigurePage Page 254254

Making Recombinant Making Recombinant DNADNA

nick

5’

3’

3’

5’

G A A T T C

C T T A A G

nick

G A A T T C

C T T A A G

DNA ligase action

In-text In-text figurefigurePage Page 254254

Using PlasmidsUsing Plasmids

Plasmid is small circle of bacterial Plasmid is small circle of bacterial

DNADNA Foreign DNA can be inserted into Foreign DNA can be inserted into

plasmidplasmid

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

Using PlasmidsUsing Plasmids

DNA fragments+enzymes

recombinantplasmids

host cells containing recombinant plasmids

Figure Figure 16.416.4

Page 255Page 255

Amplifying DNAAmplifying DNA

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

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

Polymerase Chain Polymerase Chain ReactionReaction

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

ends of single strandsends of single strands DNA polymerase uses free DNA polymerase uses free

nucleotides to create nucleotides to create complementary strandscomplementary strands

Doubles number of copies of DNADoubles number of copies of DNA

Polymerase Polymerase Chain Chain ReactionReaction

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

Figure Figure 16.616.6

Page 256Page 256

Stepped Art

Polymerase Polymerase Chain Chain ReactionReaction

Figure Figure 16.616.6

Page 256Page 256

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

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 Even full siblings can be

distinguished from one another by distinguished from one another by

this technique 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 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

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 DNA molecules are negatively

charged and move toward positive charged and move toward positive end of gelend of gel

Smaller molecules move faster than Smaller molecules move faster than larger oneslarger ones

Analyzing DNA Analyzing DNA Fingerprints Fingerprints

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

use of a radioactive probeuse of 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

Genome SequencingGenome Sequencing

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

Automated DNA sequencing now Automated DNA sequencing now main methodmain method

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

Gene LibrariesGene Libraries

Bacteria that contain Bacteria that contain

different cloned DNA different cloned DNA

fragmentsfragments

Genomic libraryGenomic library

cDNA librarycDNA library

Engineered ProteinsEngineered Proteins

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

medically valuable proteinsmedically valuable proteins

Insulin, interferon, blood-clotting Insulin, interferon, blood-clotting

factorsfactors

VaccinesVaccines

Cleaning Up the Cleaning Up the EnvironmentEnvironment

Microorganisms normally break Microorganisms normally break

down organic wastes and cycle down organic wastes and cycle

materialsmaterials

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

down pollutants or to take up larger down pollutants or to take up larger

amounts of harmful materialsamounts of harmful materials

The Ti plasmidThe Ti plasmid

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

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

foreign genein plasmid

plant cell

Figure 16.11Page 261

Engineered PlantsEngineered Plants

Cotton plants that display resistance Cotton plants that display resistance to herbicideto herbicide

Aspen plants that produce less lignin Aspen plants that produce less lignin and more celluloseand more 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

First Engineered First Engineered MammalsMammals

Experimenters used mice with Experimenters used mice with hormone deficiency that leads to hormone deficiency that leads to dwarfism 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 Nucleus from mammary gland cell

was inserted into enucleated egg was inserted into enucleated egg

Embryo implanted into surrogate Embryo implanted into surrogate

mother mother

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

from which mammary cell was takenfrom which mammary cell was taken

Designer CattleDesigner Cattle

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

Embryos can be genetically modifiedEmbryos can be genetically modified create resistance to mad cow create resistance to mad cow

diseasedisease engineer cattle to produce human engineer cattle to produce human

serum albumin for medical useserum albumin for medical use

The Human Genome The Human Genome InitiativeInitiative

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

waste of resourceswaste of resources Process accelerated when Craig Process accelerated when Craig

Ventner used bits of cDNAs as hooks Ventner used bits of cDNAs as hooks to find genesto find genes

Sequencing was completed ahead of Sequencing was completed ahead of schedule in early 2001schedule in early 2001

GenomicsGenomics

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

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

Using Human GenesUsing Human Genes

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

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

Very difficult to get modified genes Very difficult to get modified genes to work where they shouldto work where they should

Can Genetically Can Genetically Engineered Bacteria Engineered Bacteria

“Escape”?“Escape”?

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

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

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?