Copyright 2003 Pearson Education, Inc.

publishing as Benjamin Cummings

p.174

Biotechnologyuseoflivingorganismstocreateproducts
orhelpprocesses
Ex.HGH,insulin
RecombinantDNAsegmentofDNAcontaining
sequencesfromdifferentorganisms
HowisDNAmanipulated?

RestrictionenzymescutDNAatspecificsites
andcreatestickyends
G T G G
T
T
A A
A C C
C
A
A
T T

T A

C C T
C C A
G A
G G A
G G T C
T

G A
T C
G
T
G
C T C
T
T
C A G
A
A
A
C
A
C
T
G
G A
C A
A
G G
T C T
T T A
GA
GA
T
TC C
C
G
T
T G
C T
T
C G A
T T G
A
A
C C A G AA C G G
A
T TC
T
C
C
T
A
A
G
C
C
A
G
G
G
A
C
A
A
G
A
T
A
C
G G T C T T G G C
TA A G
T T

Complementaryendswillfusetoproduce
alongstrandofDNA

 The DNA is then integrated into the recipient

cells chromosome
Donated DNA

Degraded DNA
Crossovers

Recipient cells
chromosome
Figure 12.1D
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Recombinant
chromosome

PlasmidsareextraringsofDNAthatreplicateinbacteria.
DNAcanbeinsertedintoplasmids.
bacterium
bacterial
chromosome
plasmid

CloningVectors

Bacterium

Human cell

Plasmid

DNA
Human protein

Bacterial
chromosome

1. Use restriction enzymes.

2. Insert gene into plasmid.

recombinant DNA
transformation

3. Transfer the plasmid back

into bacterial cell.
replication

4. Let bacterial cells replicate.

bacterial
clones

Recombinant DNA products

seed protein for artificial snow
Insulin for diabetes treatment
Enzymes that clean up toxic waste spills
Growth Hormones (Human, Bovine)
TPA: Tissue Plasminogen Activator for
treatment of heart attacks
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

 The polymerase
chain reaction
(PCR) can
quickly clone a
small sample of
DNA in a test
tube

Initial
DNA
segment

Selection of
specific sequence
1

Number of DNA molecules

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

8
Figure 12.12

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Gel electrophoresis sorts DNA molecules by size

Restriction fragments of DNA are compared by
size
Mixture of DNA
molecules of
different sizes

Longer
molecules
Power
source

Gel
Shorter
molecules

Glass
plates
Completed gel

Figure 12.10
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

DNA
forensics

Eggmicroinjectiontoproduce
transgenicanimal

Credit: Science VU/Visuals Unlimited

Egg manipulation via microinjection.

Growbiggerfishfaster.
Salmonwithgenefromanotherfishspecies

 Uses of
transformed
animals:
Produce
medicines more
easily
Ex. sheep and gene to
treat cystic fibrosis
Goats and AT3 gene to
prevent blood clots
Figure 12.16
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Fig. 11-14, p.173

Geneticengineeringofplants
MethodstoinsertDNA:
1. Ballistics
2. Protoplasts
3. Agrobacteriumasvector
Tiplasmid

Credit: Brad Mogen

An extremely large Agrobacterium tumefaciens tumor (crown gall disease) and secondary
tumors on Kalanchoe stem.

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.
The plasmid DNA
becomes
integrated into
one of the plants
chromosomes.

c The plant cell

divides. Its
descendant
cells form an
embryo, which
may develop
into a mature
plant that can
express the
foreign gene.

A young
plant
expressing
a fluorescent
gene product

Fig. 11-12, p.171

Genetically modified crops

Golden rice with Vitamin A
Cotton resistant to boll weevil
Soybeans resistant to herbicide (Roundup)
Corn resistant to European corn borer
Rapeseed with healthier vegetable oil

Benefitsandrisks
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

HowDollywascloned

DNA
udder cells
white sheep
embryo
surrogate
mother
egg cell
black sheep

Dolly

Cloningofhumancells
Regenerativemedicine
Bone,pancreascells,skin
Stemcellsthe$6billionpromise?

Gene therapy may someday help treat a

variety of diseases
treat disease by altering an
afflicted individuals genes

Cloned gene (normal allele)

1 Insert

normal gene
into virus
Viral nucleic
acid

Ex vivo

Retrovirus

In vivo

2 Infect bone

marrow cell
with virus

Stem cells

3 Viral DNA

inserts into
chromosome
Bone marrow
cell from patient
Bone
marrow

Figure 12.19
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

4 Inject cells

into patient

HumanGenomeProject
3.2billionbasesin22autosomes+X,Y
Draftsequencecompletedin2003
Availableat

www.ornl.gov/sci/techresources/Human_Genome/
home.shtml
www.ucsc.genome.edu

Whatdoesthehumangenome
sequencetellus?
20Kto25Kgenes
99.9%alike,acrossallraces
97%ofDNAisnottranscribed
Spacersbetweengenes
Structural(centromeres,telomeres)
Regulatory(enhancers,promoters)
Leftoversofevolution?

Howarespecificgenesidentified?
1.Isolateitfromagenomiclibraryby
homologywithagenefromanother
organism.
2. FindmRNAforthegene,makecDNA
fromit.
3. MakeDNAsequencebasedonprotein
sequence.

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

1. Nucleic acid probes identify clones

carrying specific genes
A nucleic acid probe can tag a desired gene in a
library
Radioactive
probe (DNA)
Mix with singlestranded DNA from
various bacterial
(or phage) clones
Single-stranded
DNA

Base pairing
indicates the
gene of interest
Figure 12.8A
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

cDNA

mRNA

Complementary
DNA
mRNA
Using reverse
cDNA
transcriptase
Assembles DNA
on mRNA
template

reverse
transcriptase

DNA
polymerase

DNA
DNA
Fig. 11-4, p.164

DNA microarrays test for the expression of

many genes at once
A labeled probe can
reveal patterns of
gene expression in
different kinds of
cells

cDNA
DNA of gene

DNA
microarray,
actual size
(6,400 genes)

Figure 12.9
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Gene Therapy
What is it?
How is it done?
Does it work?

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Gene therapy
Goal - Treat diseases caused by mutated genes
Method - Add a normal gene or block an
abnormal gene in enough cells to restore
normal function
Target - somatic cells

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

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

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Knockout gene therapy

Goal: turn off a gene that is causing a
disorder
Strategies:
Antisense
Triple helix oligos
Spliceosome
Ribozyme
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

How is gene therapy done?

1.

Identify the gene(s) responsible for the disorder

2.

Make copies of the normal gene

3.

Insert the copies into vectors (i.e., viruses)

4.

Infect the affected cells with the vectors

5.

Activate the gene

Transcription and translation take place

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Critical factors in choosing a vector

Gene size

Limited room in vector genome

Target tissue

What cells can the vector infect?

Integration into the genome

Without integration, only short-term

effect
Random integration may disrupt other
genes

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Photo courtesy of Van de Silva

Gene Therapy Successes

Ashanti de Silva
successfully treated for
ADA deficiency - 1990

Ryes Evans successfully

treated for SCID - 2001
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Gene Therapy Problems

Jesse Gelsinger died of
complications due to an
immune system response while
participating in a clinical trial

Three children treated for SCID developed

leukemia due to disruption of a gene that
regulates cell division
Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings

Ethical and Social Issues

Patient safety while participating in
clinical trials
Which applications are therapies and
which are enhancements?
Designer babies

Access to gene therapies

Copyright 2003 Pearson Education, Inc. publishing as Benjamin Cummings