ii.4 biotechnology & rdna

8/13/2019 II.4 Biotechnology & rDNA

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Recombinant DNA

and GeneticEngineering


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Biotechnology and

Recombinant DNA Biotechnology

The use of microbiological and biochemical

techniques to solve problems and produceproduct

Recombinant DNA techniques

Methods used to manipulate DNA to intentionallygenetically alter organisms through genetic

engineering

Often to give organisms more useful traits


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Familial Hypercholesterolemia

Gene encodes protein that serves as cells

LDL receptor

Two normal alleles for the gene keep

blood level of LDLs 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 for LDL

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 of LDLs down 20 percent No evidence of atherosclerosis

Cholesterol levels remain high

Remains to be seen whether procedure willprolong her life


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Genetically Modified Organisms

(GMOs) GMOs are organisms that have had

genetic material removed and/or

inserted in order to change a

particular trait or traits of the

organism.

The process is called gene splicing

or genetic engineering

Organisms produced by

transplanting genetic materials

between different types of organisms

are called transgenic organisms.


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Transgenic Organism Examples

Genes from bacteria are spliced into corn and

cotton to make them less susceptible to insect

damage

Human growth hormone implanted into mice &

other animals so that it can be harvested

ANDi (first transgenic monkey) is a rhesus

monkey carrying GFP protein, showing foreign

gene can be inserted into primate chromosome

May lead to primate models of human diseases


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Genetic Changes

Humans have been changing the

genetics of other species for thousands

of years

Artificial selection of plants and animals

Natural processes also at work

Mutation, crossing over


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Definitions

Recombinant DNA technology:

techniques & tools used to analyze genes

Cut DNA up and recombine pieces

Amplify modified pieces

Genetic Engineering: uses the abovetechnology to isolate & modify genes in

organisms or even to insert new genes


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Genetic EngineeringRecombinant DNA (rDNA) Technology

rDNA technology

involves cloning DNA by

cutting & pasting DNAfrom different sources

Restriction enzymes &

DNA ligases are

important enzymes forthis process

DNA ligasesjoin together

adjacent DNA fragments


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Fundamental Tools of

Biotechnology Basic components of molecular biologists

toolkit

Restriction enzymes

Gel electrophoresis

DNA probes

Primers


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Restriction enzymes

Restriction enzymes are DNA-

cutting enzymes that are found in

bacteria

They are also called

endonucleases (cut within DNA

sequences)

Microbiologists from 1960sdiscovered that some bacteria are

protected from destruction by

viruses because they cut viral DNA,

restricting viral replication


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Restriction Enzymes

Molecular scissors that cut DNA at a

specific nucleotide sequence

Over 200 different restriction enzymes areknown, each isolated from bacteria and

able to cut DNA in a unique manner


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Restriction enzymes Q & A

In 1970, Hamilton Smith isolated HindIII (1st restriction

enzyme well characterized and used for DNA cloning),

which comes from Haemophilus influenzae.

They are named based on genus & species of bacteria

it was isolated from. (EcoRI = Escherichia coli, RY13).

They cut DNA by cleavingphosphodiester bonds (in

sugar-phosphate backbone) that join adjacent

nucleotides

Which was the first one well understood?

How are they named?

How do they work?


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Specificity

Restriction enzymes show specificity for certain

substrates (DNA in this case)

They recognize, bind to, and cut DNA at specific

sites called restriction sites (recognition site) Usually a 4-base pair or 6-base pair cutter

Restriction sites are palindromes (reads same

forward & backwards on opposite strands)


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Restriction cuts

Some cut DNA to create fragmentswith overhanging single-stranded ends

(sticky ends or cohesive ends),

while others create fragments with

non-overhanging ends (blunt ends)

Enzymes that create sticky ends are favoredfor cloning experiments since the

DNA fragments can be easily joined together

DNA from anysource can be digested (as long as it has the specific restriction

site)


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Restriction

enzymes


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GE Application

In 1972, Paul Berg joined

DNA from E.coliand a primate

virus called SV40

He cut both with EcoRI

(restriction enzyme)

He then added fragments totube with DNA ligase

This became 1st recombinant

DNA molecule


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Making Recombinant DNA

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

figure


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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

In-text

figure


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Recombinant DNA Technology

Cutting andPasting

Enzymes:

Restriction

enzymes =

cut

Ligase =paste


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DNA (Gene) cloning

Want to study or isolate a particular gene

Need to get many copies (amplification) of the

gene so it can be studied adequately Most organisms only have one or two copies of

any gene per cell, so we need a way to amplify

copies of that gene

Do that via cloning into a vector

This allows scientists to make additional copies

of the gene using bacteria


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Vectors

Plasmids can be used as vectors

(pieces of DNA that can accept,

carry, and replicate other pieces of

DNA)

1st plasmid vector pSC101

(SC = Stanley Cohen, pictured left)

Contained gene for tetracycline

(antibiotic) resistance and restriction

sites for several enzymes

rDNA animation
http://www.bioteach.ubc.ca/TeachingResources/Applications/GMOpkgJKloseGLampard2.swfhttp://www.bioteach.ubc.ca/TeachingResources/Applications/GMOpkgJKloseGLampard2.swf


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Vectors

Cohen & Boyer (pictured left)

awarded patents (1980) for pSC101

and gene splicing & cloning

technologies

Major concern at the time was the

thought of recombinant bacteria

leaving the lab

Boyer joined forces with Robert

Swanson (venture capitalist) to

create Genentech in an effort to

commercialize these technologies


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Vector Features

Modern plasmid DNA cloning vectors

usually consider 6 desirable features:

1. Size (must be small enough to

separate easily)

2. Origin of replication (ori) - DNA

sequence at which replication is initiated

3. Multiple cloning site (MCS) - a stretch of DNA with recognition sequences for

common restriction enzymes (Engineered into plasmid so that digestion does not result

in loss of DNA fragment)


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Vector Features

4. Selectable marker genes - allow for selection and

identification of transformed bacteria

Most common selectable markers

are antibiotic resistance.

Lac zgene widely used (gene of

interest inserted within lac zgene)

Plated on X-gal (substrate similarto lactose but turns blue when

cleaved by -gal); so, recombinant

bacteria turn blue &

nonrecombinant are white


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Using Plasmids

DNA

fragments

+

enzymesrecombinant

plasmids

host cells containing

recombinant plasmidsFigure 16.4Page 255


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Using

Plasmids


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Vector Features

5. RNA polymerase promoter

sequences - place where RNA

polymerase binds to begintranscription

6. DNA sequencing primer

sequences - known sequence

that allows sequencing ofcloned DNA fragments that have

been inserted into the plasmid


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Types of Vectors

Oneprimary limitation of bacterial plasmids as vectors is

the size of DNA fragments (usually cannot exceed 6-

7kb: 6000-7000 base pairs).

Bacteriophage vectors

Expression vectors

Bacterial artificial chromosomes (BACs)

Yeast artificial chromosomes (YACs)

Tumor-inducing (Ti) vectors


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Phage Cloning Vectors

Fragments up to 23 kb can be may be accommodatedby a phage vector

Lambda is most common phage

60% of the genome is needed for lytic pathway. Segments of the Lambda DNA is removed and a stuffer

fragment is put in.

The stuffer fragment keeps the vector at a correct size

and carries marker genes that are removed when foreignDNA is inserted into the vector.

Example: Charon 4A Lambda


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Bacterial Artificial Chromosomes(BACs) and

Yeast Artificial Chromosomes(YACs)

BACs can hold up to 300 kbs.

The F factor of E.coli is capable ofhandling large segments of DNA.

Recombinant BACs are introducedinto E.coli by electroportation ( a brief

high-voltage current). Once in thecell, the rBAC replicates like an Ffactor.

Example: pBAC108L

Has a set of regulatory genes, OriS,and repE which control F-factor

replication, and parA and parB whichlimit the number of copies to one ortwo.

A chloramphenicol resistance gene,and a cloning segment.

YACs can hold up to 500 kbs.

YACs are designed to replicate asplasmids in bacteria when no foreignDNA is present. Once a fragment isinserted, YACs are transferred to cells,they then replicate as eukaryotic

chromosomes. YACs contain: a yeast centromere, two

yeast telomeres, a bacterial origin ofreplication, and bacterial selectablemarkers.

YAC plasmidYeast chromosome

DNA is inserted to a unique restriction

site, and cleaves the plasmid with anotherrestriction endonuclease that removes afragment of DNA and causes the YAC tobecome linear. Once in the cell, the rYACreplicates as a chromosome, alsoreplicating the foreign DNA.


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Gene Transfer

Cohen discovered that plasmid DNA enters a

bacterial cell (transformation) treated with

calcium chloride, chilled on ice, then briefly

heated

A more recent transformation technique iselectroporation (brief pulse of high-voltage

electricity to create tiny holes in bacterialcell

wall allowing DNA to enter)

Cells that have been treated for transformation (so they are more receptive to

take up DNA) are called competent cells


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Biolistics

Sometimes, biolistics are used

in order to have foreign DNA enter

a cell

DNA is blasted into the cell using

tiny bullets composed of tungsten

or goldparticles with DNA attached

Done with a gene gun (akabioblaster)

Can be used on bacteria, yeasts,

& mammalian cell lines


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Polymerase Chain Reaction A faster method of amplifying or making more

copies of a gene is PCR

PCR requires short pieces of single-strandedDNA which match up to a regions at the

beginning & end of the gene to be amplified,called primers

Primers are required as a starting point for theDNA polymerase, the same enzyme used inDNA replication

DNA polymerase then makes copy after copy ofthe gene. In a couple of hours more copies canbe made by PCR than are made using bacteria &cloning vectors


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Polymerase

Chain Reaction

Double-strandedDNA to copy

DNA heated to90 94C

Primers added tobase-pair withends

Mixture cooled;base-pairing ofprimers and endsof DNA strands

DNA polymerasesassemble newDNA strands

Figure 16.6

Page 256

Stepped Art


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Polymerase

Chain Reaction

Figure 16.6

Page 256

Stepped Art

Mixture heated again;

makes all DNA

fragments unwind

Mixture cooled; base-

pairing between

primers and ends ofsingle DNA strands

DNA polymeraseaction again

doubles number of

identical DNA

fragments


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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 larger

ones


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Gel Electrophoresis

B ilding a DNA Librar and Finding a DNA Clone of Yo r Gene or Gene Prod ct


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Figure 18.7 Figure 18.8

Building a DNA Library and Finding a DNA Clone of Your Gene or Gene Product


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Making cDNAmRNA transcript

mRNAcDNA hybrid

single-stranded cDNA

double-stranded cDNAFigure 16.5

Page 255


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Confirmation of a Cloned Gene

One method used identify a specific

gene is called a Southern Blot

Steps:1. Cut DNA from bacteria with restriction

enzymes.

2. DNA fragments are separated by a gelsoaked in a chemical solution.

Gel electrophoresis uses an electric field

within a gel to separate molecules by their size


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Negatively charged DNA is put into these

wells.

They are attracted to the positive pole fromthe electric field.

The Smallest DNA fragments move the

fastest


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Gel Electrophoresis

DNA plus restriction

enzyme

Mixture of DNA

fragments

Gel

Power

source

Longer

fragments

Shorter

fragments


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3. The DNA separated is then transferred to

a filter paper (blotted) and a probe

solution is added. Probes: radioactive RNA or single-stranded

DNA pieces that are complementary to the

gene of interest

4. Only DNA fragments complementary to

the probe will form and bind bands


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DNA probes

Used to locate nucleotide sequences in DNA

or RNAProbe is single-stranded piece of DNA tagged

with detectable marker

Location can be easily determined

Probe will hybridize to complementary

fragment of interest

Fundamental Tools of Biotechnology


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Using a DNA probe to find the

colony with the gene of interestAlignment Marks


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Primers

Single stranded DNA fragments that bind

sequences of DNAUsed in in vitro DNA synthesis

Primer serves fragment for addition of DNA

nucleotides

Fundamental Tools of Biotechnology

A li ti f


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Applications of

Genetic Engineering

Genetically engineered

bacteria

Genetic engineering

relies on DNA cloning

Process ofproducing copies of

DNA

Cloned DNA

generally combined

with carrier moleculecalled cloning vector

Insures replication

of target DNA


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Genetically engineered organisms have

variety of uses

Protein productionDNA production

Researching gene function and regulation

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Applications of

Genetic Engineering
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Protein production

Produce commercially important proteins

Pharmaceutical proteins Human insulin

Vaccines

Hepatitis B vaccine

Commercially valuable proteins Chymosin An enzyme that catalyzes the coagulation of

milk used in the production of cheese

Applications of

Genetic Engineering


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DNA production

Researches interested in acquiring available

sources of specific DNA fragmentsFragments used for

DNA study

Looking genomic characteristics

DNA vaccines

Looking at injecting DNA of pathogen to produce immune

response

Applications of

Genetic Engineering

Applications of


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Applications of

Genetic Engineering Researching gene function and

regulation

Function and regulation canbe more easily study incertain bacteria

E. coliused often due toestablished protocols

Gene expression can bestudied by gene fusion

Joining gene being studiedto reporter gene

Reporter gene encodesobservable trait

Trait makes it possibleto determine changesin gene


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Genetically engineered eukaryotes Yeast serve as important eukaryotic model for gene

function and regulation

Plant or animal that receive engineered gene termedtransgenic organism

Examples of genetically altered plants include

Pest resistant plants Corn, cotton and potatoes

Herbicide resistant plants

Soybeans, cotton and corn Plants with improved nutrient value

Rice

Plants as edible vaccines Bananas and potatoes

Applications of

Genetic Engineering


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Applications of

Probe Technologies

Variety of technology employ DNA probes

Colony blotting

Southern blotting (check for specific DNA inelectrophoresis samples)

Fluorescence in situ hybridization (FISH) (checkfor specific DNA sequences in whole chromosomes)

DNA microarray

Applications of


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Applications of

Probe Technologies

Colony blotting

Used to detect specific DNA

sequences in colonies

grown in agar plates

Colonies are transferred inplace on nylon membrane

Colony blots are used to

determine which cells

contain genes of interest

Applications of


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Applications of

Probe Technologies

Southern blotting

Uses probes to detect

DNA sequences in

restriction fragments

separated using gelelectrophoresis

Application of

Southern blotting is

locating DNA

sequences similar toones being studied


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Applications of

Probe Technologies

Fluorescence in situ hybridization

(FISH)

Uses fluorescently labeled probes to detectcertain nucleotide sequences

Detects sequences inside intact cells

Specimens are viewed using fluorescence

microscopes

FISH can be used to identify specific

properties of bacteria

Mycobacterium tuberculosis in sputum sample


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Applications for

DNA Sequencing

Knowing DNA sequence of particular cell

helps identify genetic alterations

Alterations that may result in disease Sickle cell anemia

Due to single base-pair change in a gene

Cystic fibrosis

Caused by three base-pair deletion

Applications of


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Applications of

Polymerase Chain Reaction

Creates millions ofcopies of given regionof DNA in matter ofhours

Technique exploitsspecificity of primers

Allows for selectivereplication of chosenregions Termed target DNA

Large amounts ofDNA can beproduced from verysmall sample

Care must be taken to

prevent contamination with

external source of target

DNA

Basis for false-positive

test results

PCR Presentation

Techniques Used in Genetic
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Techniques Used in Genetic

Engineering

Obtaining DNA to be clonedGenerally through cell lysis

Generating a recombinant molecule

Restriction enzymes and ligases are used tocreate a recombinant molecule

Introducing recombinant molecule intonew host

Host acts as an incubator for DNAreplication

DNA-mediated transformation often used to

get DNA into host

Techniques used in Probe


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Technologies

Probe technologies include

Colony blotting

Southern blottingFISH

Microarray technology



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Techniques in colony and Southern

blotting

Blotting steps transfer sample to nylonmembrane

Probe is added

Probe hybridizes with complementary sequence

Process is used to locate positions of

hybridized probe


Technologies



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Techniques used in FISH

Sample preparation is critical

Methods used depend on type of organismSpecimen is applied to glass slide

Fluorescent label is applied and incubated

Incubation allows for hybridization

Specimen is view with fluorescence

microscope


Technologies


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Techniques Used in DNA

Sequencing Dideoxychain termination

Elements for termination reaction include Single-stranded DNA template

Primer that anneal to template DNA polymerase

Each of the nucleotide bases One of these bases is labeled with marker for detection

Dideoxynucleotides Like deoxynucleotide counterparts but lack 3 OH

Incorporation causes chain termination

Special gel electrophoresis used to separateDNA fragments by size



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Sequencing

Automated DNA sequencing

Most automated systems

use fluorescent dyes to

detect newly synthesized

DNA

Gel electrophoresis used

to separate fragments

into colored bands

Laser used to detect

color differences

Order of color reflects

nucleotide sequence

Techniques Used in Polymerase Chain


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Techniques Used in Polymerase Chain

Reaction

Starting with double stranded DNA molecule, process

involves number of amplification cycles

PCR requires three step amplification cycle

Step 1: double stranded DNA denatured by heat

Step 2: primers anneal to complementary sequence of

target DNA and DNA synthesis occurs with heat stable

DNA polymerase

Step 3: duplication of target DNA DNA is amplified exponentially


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Confirmation of Cloned Genes

Why do this?

Bacterial colonies can be used to produce

large quantities of the protein (used to studyor make drugs)

Benefits of Recombinant


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Benefits of Recombinant

Bacteria

1. Bacteria can make human insulin or

human growth hormone.

1. Bacteria can be engineered to eat oil

spills.


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Engineered Proteins

Bacteria can be used to grow medically

valuable proteins

Insulin, interferon, blood-clotting factors

Vaccines


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Creating HGH

Human Cell

Gene for human

growth hormone

Recombinant

DNA

Gene for human

growth hormone

Sticky

ends

DNA

recombination

DNAinsertion

Bacterial Cell

Plasmid

Bacterial

chromosome

Bacterial cell for

containing gene for

human growth hormone

The DNA of plants and animals


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The DNA of plants and animals

can also be altered.

PLANTS

1. disease-resistant andinsect-resistant crops

2. Hardier fruit

3. 70-75% of food insupermarket isgenetically modified.


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Engineered Plants

Cotton plants that display resistance to

herbicide

Aspen plants that produce less lignin andmore cellulose

Tobacco plants that produce human

proteins Mustard plant cells that produce

biodegradable plastic

How to Create a Genetically


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How to Create a Genetically

Modified Plant

1.Create recombinantbacteria with desiredgene.

2. Allow the bacteria toinfect" the plant cells.

3. Desired gene isinserted into plantchromosomes.

Pl t T f ti


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Plant Transformation

Recombinant

plasmid

Gene to be

transferred

Agrobacterium

tumefaciens

Cellular

DNA

Transformed bacteria introduce

plasmids into plant cells

Plant cell colonies

Complete plant is

generated from

transformed cell

Inside plant cell,

Agrobacterium inserts part of

its DNA into host cellchromosome

What do you think about eating


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What do you think about eating

genetically modified foods?

Genetically modified organisms are


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Genetically modified organisms are

called transgenic organisms.

TRANSGENIC ANIMALS

1. Mice used to study humanimmune system

2. Chickens more resistant toinfections

3. Cows increase milk supplyand leaner meat

4. Goats, sheep and pigsproduce human proteins intheir milk

Transgenic Goat


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Human DNA in

a Goat Cell

This goat contains a humangene that codes for a blood

clotting agent. The blood

clotting agent can be harvested

in the goats milk.

.

Transgenic Goat


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Cloning Dolly

1997 - A sheep cloned from an adult cell

Nucleus from mammary gland cell was

inserted into enucleated egg

Embryo implanted into surrogate

mother

Sheep is genetic replica of animal from

which mammary cell was taken


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CloningA donor cell is taken from

a sheeps udder.

DonorNucleus

These two cells are fused

using an electric shock.

Fused Cell

The fused cell

begins dividing

normally.

Embryo

The embryo is placedin the uterus of a foster

mother.Foster

Mother

The embryodevelops normallyinto a lambDolly

Cloned Lamb

Egg Cell

An egg cell is taken

from an adult

female sheep.

The nucleus of theegg cell is removed.

How to Create a


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Desired DNA is

added to an egg cell.

Transgenic Animal

H H H !


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Ha Ha Ha!

Genetic Engineering and


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g g

Crime Scenes

What are these techniques


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What are these techniques

used for?

Forensic: identifying criminals & victims

Identifying disease genes in animals &

humans Gene Therapy: inserting of new working

copies of genes into humans

Animal knockouts: turning off of a specificgene in order to discover its function


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DNA Fingerprinting

Gene Therapy


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Gene Therapy

Figure 19-20 Figure 19-21


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The Human Genome Initiative

Goal - 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 mostly completed aheadof schedule in early 2001


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Ethical Issues

Who decides what should be

corrected through genetic

engineering?

Should animals be modified to provide

organs for human transplants? Should humans be cloned?

National Institutes of Health


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National Institutes of Health

(NIH) Concerns arose

because of new

techniques In 1975, NIH formed

the Recombinant DNA

Advisory Committee

(RAC) to evaluate risksand establish guidelines

for rDNA technology

ii.4 biotechnology & rdna

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