Genetic Modification and

Biotechnology

Topic 3.5

Manipulating the Genome

Genetic Engineering involves cleaving DNA

into small fragments and inserting the

fragments into another organism.
Used in medicine, agriculture,
industry, research

Big Questions:
1) What is the relationship between science
and technology?
2) What are the implications of genetic
technologies for society?
3) What ethical and moral responsibilities
do scientists have?

Restriction Enzymes
Gene cloning and genetic engineering
were made possible by the discovery of
restriction endonucleases that cut DNA
molecules at specific locations.

Restriction Enzymes
In nature, bacteria use restriction
enzymes to cut foreign DNA, such as
from phages or other bacteria.
DNA scissors
Hundreds have been isolated and named
after bacteria they were isolated from

Restriction Enzymes
Restriction enzymes cleave [cut] both
strands of the DNA molecule at certain
points called palindromes.
"Was it a rat I saw?"
"Step on no pets"
"Dammit, I'm mad!

Race car
Taco cat
Evil olive
Never odd or even

Useless knowledge:
Aibohphobia- the fear
of palindromes

Restriction Enzymes
Restriction enzymes cleave [cut] both
strands of the DNA molecule at certain
points called palindromes.

EcoRI recognizes GAATTC

segments & cuts them between
the G & A
cut

cut

This produces sticky ends that will

readily bond to their sticky compliments.

G
C
T T

G
T C A

A A

G C

A A

A A

Foreign DNA
cut with
the same
restriction
enzyme

G
C
T T

G A C T T

G C T T

A A

G C
T T

A A

G A C T T

A A

G C
T T

A A

Forms recombinant DNA

This is
known as

Splicing

Restriction Enzymes
Because the target sequence usually
occurs (by chance) many times on a
long DNA molecule, an enzyme will make
many cuts.
Copies of a DNA molecule will always
yield the same set of restriction
fragments when exposed to a specific
restriction enzyme.

Vectors
Recombinant plasmids are produced by
splicing restriction fragments from
foreign DNA into plasmids.
These can be returned relatively
easily to bacteria.

Vectors
The original plasmid used to produce
recombinant DNA is called a cloning
vector, which is a DNA molecule that
can carry foreign DNA into a cell and
replicate there.

Then, as a bacterium carrying a

recombinant plasmid reproduces, the
plasmid replicates within it.
Bacteria are most commonly used as
host cells for gene cloning because
DNA can be easily isolated and
reintroduced into their cells.

Bacteria cultures also grow quickly,

rapidly replicating the foreign
genes.

Cloning
The process of cloning a human gene in
a bacterial plasmid can be divided
into four steps.

Cloning
1. Isolation of vector and gene-source DNA.
The source DNA comes from human tissue
cells.
The source of the
typically

plasmid is
E. coli.

This plasmid carries an

ampR gene,
conferring
resistance to the
antibiotic ampicillin.

Cloning
2. Insertion of DNA into the vector
By digesting both the plasmid and human DNA
with the same restriction enzyme we can
create thousands of
human DNA
fragments,
one fragment with the
gene that we want, and
with compatible sticky ends on
bacterial plasmids.

Cloning
2. Insertion of DNA into the vector
After mixing, the human fragments and cut
plasmids form complementary pairs that are
then joined by
DNA ligase.
This creates a
recombinant DNA
molecules.

mixture of

Cloning
3. Introduction of the cloning vector into
cells.
Bacterial cells take up the recombinant
plasmids by transformation.
This creates a diverse pool of bacteria, some
bacteria that have taken up the desired
recombinant plasmid DNA, and bacteria that lack
the plasmid

Cloning
4. Cloning of cells
(and foreign genes)
We can plate out the
transformed bacteria
on solid nutrient
medium containing
ampicillin. Only
bacteria that have the
ampicillin-resistance
plasmid will grow.

Eukaryotic Engineering
More difficult that prokaryotic
engineering due to a nuclear membrane
Requires different strategies

Eukaryotic Engineering

Eukaryotic Engineering

Polymersase Chain Reaction (PCR)

This technique can quickly amplify any
piece of DNA without using cells.

 The DNA is
incubated in
a test tube
with special
DNA
polymerase,
a supply of
nucleotides,
and short
pieces of
singlestranded DNA
as a primer

 PCR can make

billions of
copies of a
targeted DNA
segment in a
few hours.
This is
faster than
cloning via
recombinant
bacteria.

 In PCR, a
three-step
cycle:
heating,
cooling, and
replication,
brings about
a chain
reaction
that
produces an
exponentiall
y growing
population
of DNA
molecules.

 The key to easy PCR automation was the

discovery of an unusual DNA
polymerase, isolated from bacteria
living in hot springs, which can
withstand the heat needed to separate
the DNA strands at the start of each
cycle.

 PCR has amplified DNA from a variety of

sources:
Fragments of ancient DNA from a 40,000year-old frozen wooly mammoth.
DNA from tiny amount of blood or semen
found at the scenes of violent crimes.

 PCR has amplified DNA from a variety of

sources:
DNA from single embryonic cells for rapid prenatal
diagnosis of genetic disorders

 PCR has amplified DNA from a variety of

sources:
DNA of viral genes from cells infected with
difficult-to-detect viruses such as HIV

WARNING!
OLD SLIDES AHEAD

Comparisonsamongwholesetsofgenesand
theirinteractionsisthefieldofGenomics.
Oneindirectmethodofrapidlyanalyzingand
comparinggenomesisGelElectrophoresis.

Gelelectrophoresisseparatesmacromolecules
nucleicacidsorproteinsonthebasisof
theirrateofmovementthroughagelinan
electricalfield.

*Rateofmovementdepends
onsize,electricalcharge,and
otherphysicalpropertiesof
themacromolecules.

ForlinearDNAmolecules,separation
dependsmainlyonsize(lengthoffragment)
withlongerfragmentsmigratinglessalong
thegel.

Gel Electrophoresis
[in a nut shell]

suspects
EV12345

Evidence

EV12345

Who's Guilty?

Restrictionfragmentanalysisindirectly
detectscertaindifferencesinDNAnucleotide
sequences.
*AftertreatinglongDNAmoleculeswitha
restrictionenzyme,thefragmentscanbe
separatedbysizeviagelelectrophoresis.

Restrictionfragmentanalysisindirectly
detectscertaindifferencesinDNAnucleotide
sequences.
*Thisproducesaseriesofbandsthatare
characteristicofthenucleotidesequence
andthatrestrictionenzyme.

Techniquesforgenemanipulationholdgreat
potentialfortreatingdiseasebygenetherapy.
*Thisaltersanafflictedindividualsgenes.
*Anormalalleleisinsertedintosomatic
cellsofatissueaffectedbyagenetic
disorderviaavector[suchasavirus].

ViralDNAisgenetically
engineeredtoincludethe
normalgeneindividuals
withCysticfibrosis
lack.

Whenthevirusattacks
lungcells,itinjectsthe
engineeredDNA
directlyintothelung
cells

transformingthe
lungcellsDNAto
producethenormal
typeofmucus.

Bonemarrowcells,whichincludethestem
cellsthatgiverisetobloodandimmune
systemcells,areprimecandidatesforgene
therapy.
*Anormalallelecouldbeinsertedbyaviral
vectorintosomebonemarrowcells
removedfromthepatient.
*Iftheproceduresucceeds,thereturned
modifiedcellswillmultiplythroughoutthe
patientslifeandexpressthenormalgene,
providingmissingproteins.

DNAtechnologyhasbeenusedtocreate
manyusefulpharmaceuticals,mostly
proteins.
Bytransferringthegeneforaproteinintoa
hostthatiseasilygrowninculture,onecan
producelargequantitiesofnormallyrare
proteins.

Oneofthefirstpracticalapplicationsofgene
splicingwastheproductionofmammalian
hormonesandothermammalianregulatory
proteinsinbacteria.
*Theseincludehumaninsulinandgrowth
factor.

*Humaninsulin,producedby
bacteria,issuperiorforthecontrol
ofdiabetesthantheoldertreatment
ofpigorcattleinsulin.

*Humangrowthhormonebenefits
childrenwithhypopituitarism,a
formofdwarfism.

DNAfingerprintscanbeusedforensicallyto
presentevidencetojuriesinmurdertrials.

ThisautoradiographofRFLPbandsof
samplesfromamurdervictim,thedefendant,
andthedefendantsclothesisconsistentwith
theconclusionthatthebloodontheclothesis
fromthevictim,notthedefendant.

TheforensicsuseofDNAfingerprinting
extendsbeyondviolentcrimes.
*Forinstance,DNAfingerprinting
canbeusedtosettleconclusivelya
questionofpaternity.

Increasingly,geneticengineeringisbeing
appliedtoenvironmentalwork.
Scientistsareengineeringthemetabolismof
microorganismstohelpcopewithsome
environmentalproblems.

*Forexamplegeneticallyengineered
microbesthatcanextractheavymetalsfrom
theirenvironmentsand
incorporatethemetalsinto
recoverablecompounds
maybecomeimportant
bothinminingmaterials
andcleaninguphighly
toxicminingwastes.

*Inadditiontothenormalmicrobesthat
participateinsewagetreatment,new
microbesthatcandegradeotherharmful
compoundsarebeingengineered.

*Geneticallyengineeredmicrobesarebeing
usedtodegradeoilspilledintotheoceans.

Formanyyearsscientistshavebeenusing
DNAtechnologytoimproveagricultural
productivity.
*DNAtechnologyis
nowroutinelyusedto
makevaccinesand
growthhormonesfor
farmanimals.

Todevelopatransgenicorganism,scientists
removeovafromafemaleandfertilizethem
invitro.
*Thedesiredgenefromanotherorganism
areclonedandtheninsertedintothenuclei
oftheeggs.
*Somecellswillintegratetheforeign
DNAintotheirgenomesandareableto
expressitsprotein.

Todevelopatransgenicorganism,scientists
removeovafromafemaleandfertilizethem
invitro.
*Theengineeredeggsarethensurgically
implantedinasurrogatemother.
*Ifdevelopmentissuccessful,theresults
isatransgenicanimal,containinga
genesfromathirdparent,evenfrom
anotherspecies.

ThepGLOgene
thatcodesfora
proteinthat
givesthemthe
abilitytoglow
wasinserted
intoarabbits
ova.

Theovawas
implantedintoa
surrogate
motherand
voila
aglowing
bunnywas
born.

Fluorescent Fish

Agriculturalscientistshaveengineereda
numberofcropplantswithgenesfor
desirabletraits.
*Theseincludedelayedripeningand
resistancetospoilageanddisease.
*Becauseasingletransgenicplantcellcan
begrowninculturetogenerateanadult
plant,plantsareeasiertoengineerthan
mostanimals.

Geneticengineeringisquicklyreplacing
traditionalplantbreedingprograms.
*Inthepastfewyears,roughlyhalfofthe
soybeansandcorninAmericahavebeen
grownfromgeneticallymodifiedseeds.
*Theseplantsmayreceivegenesfor
resistancetoweedkillingherbicidesorto
infectiousmicrobesandpestinsects.

Round-up Ready Seeds

Scientistsareusinggenetransfertoimprove
thenutritionalvalueofcropplants.
*Forexample,atransgenicriceplanthas
beendevelopedthatproducesyellowgrains
containingbetacarotenewhichhumans
usetoproduce
VitaminA.

Scientistsareusinggenetransfertoimprove
thenutritionalvalueofcropplants.
*Currently,70%ofchildrenundertheage
of5inSoutheastAsiaaredeficientin
vitaminA,leadingtovisionimpairment
andincreased
diseaserates.

Review
Polymerase chain reaction (PCR) is a technique used on small
quantities of DNA (from a crime scene, for example) to make millions
of copies so that the sample can be analysed.
DNA profiling is a technique used to identify the origin of a sample of
DNA by using gel electrophoresis to match up fragments of the
unknown DNA with DNA that has already been identified.
The Human Genome Project has succeeded in using DNA
sequencers to make a map of all the nitrogenous bases that make
up the 46 human chromosomes: this will allow researchers to locate
base sequences that might be responsible for genetic diseases,
which might then code for beneficial molecules that could be used as
medications in the future, or which are shared by different
populations thus showing ancestries and migration patterns.

Review
In recent decades, scientists have developed laboratory
techniques to cut, copy, and paste genes to engineer bacteria,
plants, and animals with desirable genetic traits; this is the case
with genetically modified Escherichia coli bacteria used to
produce human insulin.
There are natural and artificial techniques of cloning. Binary
fission, budding, and asexual reproduction in plants using
techniques such as rooting stem cuttings, result in new individuals
that are identical to their parent. Artificial cloning includes
reproductive cloning (making a copy of an entire organism) and
therapeutic cloning (making copies of certain cells). These
laboratory techniques have something in common with genetically
engineered organisms: they carry challenging ethical
considerations that no previous generation has had to face.

Bacterial Colony Transformation

using the pGLO plasmid.

It is a fairly simple procedure

Ampicillin sensitive
E. coli cells are
transferred to cold
calcium chloride
[CaCl2].

It is a fairly simple procedure

The pGLO plasmids [which carry

genes for bioluminescence & ampicillin
resistance] are added.
pGLO

It is a fairly simple procedure

The bacterial cells are heat shocked

at 42C.
This creates small
openings in the
bacterias plasma
membrane.

It is a fairly simple procedure

Some of the
competent cells
take up the
pGLO plasmid &
are transformed.

It is a fairly simple procedure

The cells are

cold-shocked to
reestablish the
bacterias plasma
membrane.

It is a fairly simple procedure

The treated cells

are spread on agar
plates. One plate
contains ampicillin.

It is a fairly simple procedure

Only the cells that took up the pGLO

plasmid will grow
on ampicillin agar.

It is a fairly simple procedure

They will also

glow under a black
light.

Colonies of
bacteria

All producing
the proteins

"Transformed"