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Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174


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Biotechnology- use of living organisms to create productsor help processes

Ex. HGH, insulin

Recombinant DNA- segment of DNA containing

sequences from different organisms

How is DNA manipulated?


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A A

A AA

A

AA

A A

A AA A

A

A

AA

A A

A A

A AA A

A

AA A A

AA A A

A

A

A A

T T

T T TT

T T

T T

T T

TT T

T

T

T

T T

T T

T TT

TT

T

T

T

T T

T T

T

T

T

T T

C

GCG

C

G

C

GC

G

CG

C

G

C

G

C

G

C

G

C

G

G

C C

G CG

CG C

G

C

G

C

G

C

G

G G

C

CC

C

G G C

G CG

GC

G

C

G

C

G

GG G G

G

G

C C C

CC

Restriction enzymescut DNA at specific sites

and create sticky ends

Complementary ends will fuse to produce

a long strand of DNA


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Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

The DNA is then integrated into the recipientcells chromosome

igure !2.!"

Donated DNA

Recipient cellschromosome

Crossovers

Degraded DNA

Recombinantchromosome


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bacterium

bacterial

chromosome

plasmid

Plasmidsare extra rings of DNA that replicate in bacteria.

DNA can be inserted into plasmids.


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Cloning Vectors


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1. Use restriction enzymes.

2. Insert gene into plasmid.

3. Transfer the plasmid bac

into bacterial cell.

!. "et bacterial cells replicate.

bacterial

clones

replication

transformation

recombinant #$A

%lasmid

&acterium'uman cell

#$A

'uman protein &acterialchromosome


8/38Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Recombinant DNA products

seed protein for artificial snow

Insulin for diabetes treatment Enzmes that clean up to!ic waste spills

"rowth #ormones $#uman% &o'ine(

T)A* Tissue )lasminogen Acti'ator fortreatment of heart attac+s



The polymerasechain reaction$),-( can.uic+l clone asmall sample ofDNA in a testtube

/election ofspecific se.uence

igure !2.!2

Initial

DNA

segment

1 2 4 8

Nmber o! DNA molecles



-estriction fragments of DNA are compared bsize

Gel electrophoresis sorts DNA molecules by size

igure !2.!0

"i#tre o! DNA

molecles o!

di!!erent si$es

%o&er

sorce'el

'lass

plates

(onger

molecles

)horter

molecles

Completed gel


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DNA

forensics


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Egg manipulation via microinjection.

Credit: Science VU/Visuals Unlimited

Egg microinjection to produce

transgenic animal


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Grow bigger fish faster.

Salmon with gene from another fish species


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0ses oftransformedanimals*

)roduce

medicines moreeasil

E!1 sheep and gene totreat cstic fibrosis

"oats and AT2 gene topre'ent blood clots

igure !2.!#


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Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings *ig. 11+14, p.17-


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An extremely large Agrobacterium tumeaciens tumor !cro"n gall disease# and secondarytumors on $alanc%oe stem.

Credit: &rad 'ogen

Genetic engineering of plants

Methods to insert DNA:

1. Ballistics

2. Protoplasts

3. Agrobacterium as vector- Ti plasmid


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aA bacterialcell contains

a i plasmid

/purple0 that

has a !oreign

gene /blue0.

bhe bacterimin!ects a plant

and trans!ers the

i plasmid into it.

he plasmid DNA

becomes

integrated intoone o! the plants

chromosomes.

che plant celldivides. Its

descendant

cells !orm an

embro, &hich

ma develop

into a matreplant that can

e#press the

!oreign gene.

A ong

plant

e#pressinga !lorescent

gene prodct

*ig. 11+12, p.171


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Genetically modified crops

"olden rice with 3itamin A

,otton resistant to boll wee'il

/obeans resistant to herbicide $-oundup(

,orn resistant to European corn borer

-apeseed with healthier 'egetable oil

Benefits and risks


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(hite sheep

blac sheep

egg cell

udder cells

#$A

embryo

#ollysurrogate

mother

How Dolly was cloned


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Cloning of human cells

Regenerative medicine

Bone, pancreas cells, skin

Stem cells - the $6 billion promise?


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treat disease b altering anafflicted indi'iduals genes

4 E! 'i'o

4 In 'i'o

4 /tem cells

Gene therapy may someday help treat avariety of diseases

igure !2.!$

Cloned gene /normal allele0

1 Insert

normal gene

into virs

iral ncleic

acid

Retrovirs

2 In!ect bone

marro& cell

&ith virs

- iral DNA

inserts into

chromosome

3one marro&cell !rom patient

3one

marro&

4 Inect cells

into patient


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Human Genome Project

3.2 billion bases in 22 autosomes + X, Y

Draft sequence completed in 2003

Available atwww.ornl.gov/sci/techresources/Human_Genome/

home.shtml

www.ucsc.genome.edu


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What does the human genome

sequence tell us? 20 K to 25 K genes

99.9% alike, across all races

97% of DNA is not transcribed

- Spacers between genes

- Structural (centromeres, telomeres)

- Regulatory (enhancers, promoters)

- Leftovers of evolution?


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How are specific genes identified?

1. Isolate it from a genomic library byhomology with a gene from another

organism.

2. Find mRNA for the gene, make cDNAfrom it.

3. Make DNA sequence based on protein

sequence.


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1. Nucleic acid probes identify clonescarrying specific genes

igure !2.%&

Radioactive

probe /DNA0"i# &ith single+stranded DNA !romvarios bacterial/or phage0 clones

)ingle+strandedDNA

3ase pairingindicates thegene o! interest

A nucleic acid probe can tag a desired gene in alibrar


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*ig. 11+4, p.154

mRNA

mRNA

cDNA

DNA

DNA

reversetranscriptase

DNA

polmerase

cDNA

( Complementary

)*A

( Using reversetranscriptase

( Assembles )*A

on m+*A

template


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A labeled probe canre'eal patterns ofgene e!pression indifferent +inds ofcells

DNA microarrays test for the expression ofmany genes at once

igure !2.$

cDNA

DNA o! gene

DNAmicroarra,

actal si$e

/5,466 genes0


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Gene Therapy

What is it?

How is it done? Does it work?


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Gene therapy

"oal 5 Treat diseases caused b mutated genes

6ethod 5 Add a normal gene or bloc+ anabnormal gene in enough cells to restorenormal function

Target 5 somatic cells


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Which disorders are candidates for gene therapy treatment?

Disorders due to mutations in one or more genes

The responsible gene is known

The affected tissues are known and accessible


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Knocout gene therapy

Goal: turn off a gene that is causing adisorder

Strategies:

!ntisense

Triple heli" oligos

Spliceosome

#ibo$yme


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How is gene therapy done?

71 Identif the gene$s( responsible for the disorder

81 6a+e copies of the normal gene

21 Insert the copies into 'ectors $i1e1% 'iruses(

91 Infect the affected cells with the 'ectors

:1 Acti'ate the gene

4 Transcription and translation ta+e place


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Critical 'actors in choosing a (ector

"ene size

4 ;imited room in 'ector genome

Target tissue

4


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)ene *herapy +uccesses

Ashanti de /il'asuccessfull treated forADA deficienc 5 7>>?

+yes Evans successully

treated or SC,) - 0

1%

otocourtesyoVandeSilva


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)ene *herapy Problems

@esse "elsinger died ofcomplications due to animmune sstem response while

participating in a clinical trial

2%ree c%ildren treated or SC,) developed

leu3emia due to disruption o a gene t%at

regulates cell division


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Ethical and +ocial Issues

)atient safet while participating inclinical trials

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