Download - Ch15 Lecture L13

8/6/2019 Ch15 Lecture L13

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Recombinant DNA and

Genetic Engineering

Chapter 15


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Lipoproteins

Form when certain blood proteins

combine with cholesterol High-density lipoproteins (HDLs)

Low-density lipoproteins (LDLs)


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Familial Hypercholesterolemia Gene encodes protein that serves as

cellsLDL

receptor Two normal alleles for the gene keep

blood level ofLDLs low

Two mutated alleles lead to abnormally

high cholesterol levels & heart disease


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Example of Gene Therapy Woman with familial hypercholesterolemia

Part of her liver was removed

Virus used to insert normal gene forLDL

receptor into cultured liver cells

Modified liver cells placed back in patient


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Results of Gene Therapy

Modified cells alive in womans liver

Blood levels ofLDLs down 20 percent No evidence of atherosclerosis

Cholesterol levels remain high

Remains to be seen whether procedure will

prolong her life


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Genetic Changes Humans have been changing the

genetics of other species for thousandsof years

Artificial selection of plants and animals

Natural processes also at work

Mutation, crossing over


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Genetic Engineering Genes are isolated, modified, and

inserted into an organism

Made possible by recombinant

technology

Cut DNA up and recombine pieces

Amplify modified pieces


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Discovery of Restriction

Enzymes

Hamilton Smith was studying how

Haemophilus influenzae defend

themselves from bacteriophage attack

Discovered bacteria have an enzyme

that chops up viral DNA


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Specificity of Cuts

Restriction enzymes cut DNA at a

specific sequence

Number of cuts made in DNA will

depend on number of times the targetsequence occurs


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Making Recombinant DNA

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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


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Making Recombinant DNA

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


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Using Plasmids

Plasmid is small circle of bacterial DNA

Foreign DNA can be inserted into

plasmid

Forms recombinant plasmids

Plasmid is a cloning vector

Can be used to deliver DNA into another

cell


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Using Plasmids

DNA

fragments

+

enzymesrecombinant

plasmids

host cells containing

recombinant plasmids


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Making cDNA

mRNA transcript

mRNAcDNA hybrid

single-stranded cDNA

double-stranded cDNA


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Amplifying DNA Fragments can be inserted into

fast-growing microorganisms

Polymerase chain reaction (PCR)


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Polymerase Chain Reaction

Sequence to be copied is heated

Primers are added and bind to ends ofsingle strands

DNA polymerase uses free nucleotides

to create complementary strands Doubles number of copies of DNA


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Polymerase Chain Reaction

DNA is heated

Primers are added

DNA to be amplified


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Polymerase Chain ReactionMixture cools

Base pairing occurs

Complementary

strand synthesized


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Primers Short sequences that DNA polymerase

recognizes as start tags

To carry out PCR, must first determine

nucleotide sequences just before and after

the gene to be copied

Complementary primers are then created


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The DNA Polymerase

Most DNA polymerase is denatured at

high temperature

Polymerase used in PCR is from

bacteria that live in hot springs


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Temperature Cycles

DNA is heated to unwind strands

Cooled to allow base-pairing withprimers and complementary strand

synthesis

DNA is heated again to unwind strands

Cycle is repeated over and over again


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DNA Fingerprints Unique array of DNA fragments

Inherited from parents in Mendelianfashion

Even full siblings can be distinguished

from one another by this technique


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Tandem Repeats Short regions of DNA that differ

substantially among people

Many sites in genome where tandem

repeats occur

Each person carries a uniquecombination of repeat numbers


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RFLPs

Restriction fragment length polymorphisms

DNA from areas with tandem repeats is cutwith restriction enzymes

Because of the variation in the amount of

repeated DNA, the restriction fragmentsvary in size

Variation is detected by gel electrophoresis


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Gel Electrophoresis DNA is placed at one end of a gel

A current is applied to the gel DNA molecules are negatively charged

and move toward positive end of gel

Smaller molecules move faster than largerones


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Analyzing DNA Fingerprints DNA is stained or made visible by use

of a radioactive probe

Pattern of bands is used to:

Identify or rule out criminal suspects

Determine paternity


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Genome Sequencing 1995 - Sequence of bacterium

Haemophilus influenzae determined

Automated DNA sequencing now main

method

3.2 billion nucleotides in human genome

determined in this way


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Nucleotides for Sequencing Standard nucleotides (A,T,C, G)

Modified versions of these nucleotides Labeled so they fluoresce

Structurally different so that they stop DNA

synthesis when they are added to a strand


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Reaction Mixture

Copies of DNA to be sequenced

Primer

DNA polymerase

Standard nucleotides

Modified nucleotides


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Reactions Proceed Nucleotides are assembled to create

complementary strands

When a modified nucleotide is included,

synthesis stops

Result is millions of tagged copies of

varying length


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Recording

theSequence

T C C A T G G A C C

T 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

electrophoresis

gel

one of the many

fragments ofDNA migrating

through the gel

one of the DNA fragments

passing 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


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GeneLibraries

Bacteria that contain different

cloned DNA fragments Genomic library

cDNA library


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Using a Probe to Find a Gene

You want to find which bacteria in a

library contain a specific gene

Need a probe for that gene

A radioisotope-labeled piece of DNA

It will base-pair with the gene of interest


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Use of a

Probe

Colonies on plate

Cells adhere

to filter

Cells are lysed;

DNA sticksto filter

Probe is

added

Location where probe binds forms

dark spot on

film, indicates

colony with

gene


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CleaningU

p the Environment

Microorganisms normally break down

organic wastes and cycle materials

Some can be engineered to break down

pollutants or to take up larger amounts

of harmful materials

Break down oil, sponge up heavy metals


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Basic ResearchRecombinant DNA technology allows

researchers to:

Investigate basic genetic processes

Reconstruct lifes evolutionary history

Devise counterattacks against rapidly

mutating pathogens


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The Ti plasmid Researchers

replace tumor-

causing geneswith beneficialgenes

Plasmidtransfersthese genes tocultured plant

cells

foreign gene

in plasmid

plant cell


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First Engineered Mammals Experimenters used mice with hormone

deficiency that leads to dwarfism

Fertilized mouse eggs were injectedwith gene for rat growth hormone

Gene was integrated into mouse DNA

Engineered mice were 1-1/2 timeslarger than unmodified littermates


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More Mouse Modifications Experiments showed that human growth

hormone genes can be expressed in

mice Human genes are inserted into mice to

study molecular basis of genetic

disorders, such as Alzheimers disease Variety of methods used to introduce

genes


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Designer Cattle Genetically identical cattle embryos can

be grown in culture

Embryos can be genetically modified

Experimenters are attempting to create

resistance to mad cow disease

Others are attempting to engineer cattle toproduce human serum albumin for medical

use


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The Human Genome InitiativeGoal - Map the entire human genome

Initially thought by many to be a waste of

resources

Process accelerated when Craig Ventner

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

schedule in early 2001


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U

sing Human Genes Even with gene in hand it is difficult to

manipulate it to advantage

Viruses usually used to insert genesinto cultured human cells but procedure

has problems

Very difficult to get modified genes towork where they should


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Eugenic Engineering

Selecting desirable human traits

Who decides what is desirable?

40 percent of Americans say gene

therapy to make a child smarter or

better looking would be OK


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Where Do We Go Now? Can we bring about beneficial changes

without harming ourselves or the

environment? Gene therapy is not harmless

A young man died after gene therapy that

used an adenovirus

Gene therapy can save lives

Infants with disabled immune systems are

now healthy


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Can Genetically Engineered

Bacteria Escape?

Genetically engineered bacteria are

designed so that they cannot surviveoutside lab

Genes are included that will be turned

on in outside environment, triggeringdeath


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Effects of Engineered

Organisms

Opposition to any modified organisms

What if engineered genes escape into

other species?

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