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Genetic EngineeringRecombinant DNA (rDNA) Technology

Genetic EngineeringRecombinant DNA (rDNA) Technology

rDNA technology

involves cloning DNA by

cutting & pasting DNAfrom different sources

Restriction enzymes &

DNA ligases are

important enzymes for this process

DNA ligases join together

adjacent DNA fragments

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Genetically Modified Organisms

(GMOs)

Genetically Modified Organisms

(GMOs) GMOs are organisms that have had

genetic material removed and/or

inserted in order to change aparticular 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 ExamplesTransgenic 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 arhesus 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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Restriction enzymesRestriction enzymes Restriction enzymes are DNA-

cutting enzymes that are found in

bacteri a

They are also called

endonucleases (cut w ithin 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 Q & ARestriction enzymes Q & A

In 1970, Hamilton Smith isolated HindIII (1st restriction

enzyme well characterized and used for DNA cloning),

which comes from H aemophilus influenz ae.

They are named based on genus & species of bacteria

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

They cut DNA by cleaving phosphodiester 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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SpecificitySpecificity Restriction enzymes show specificity

for certain substrates (DNA in this case)

They recognize, bind to, and cut DNAat 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 cutsRestriction cuts

Some cut DNA to create

fragments with 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 f av ored for cloning

experiments since the DNA fragments can be easily joined

together

DNA from any source can be digested (as long as it has the

specific restriction site)

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GE ApplicationGE Application

In 1972, Paul Berg joined

DNA from E.coli and 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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PlasmidsPlasmids Plasmid DNA is circular

form of self-replicating DNA

that scientists canmanipulate to carry and

clone other pieces of DNA

Found primarily in

bacteria

Considered extrachromosomal DNA because they are

present in addition to chromosomes

They are small (~1000 - 1400 base pairs) in size

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VectorsVectors 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 tetr acycline

(antibiotic) resistance and restriction

sites for several enzymes

rDNA animation

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VectorsVectors Cohen & Boyer (pictured left)

awarded patents (1980) for pSC101

and gene splicing & cloningtechnologies

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 FeaturesVector 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 FeaturesVector Features4. Selectable marker genes - allow for selection and

identification of transformed bacteria

Most common selectable markersare antibiotic resistance.

Lac z gene widely used (gene of

interest inserted within l ac z gene)

Plated on X-gal (substrate similar

to lactose but turns blue when

cleaved by ß-gal); so, recombinant

bacteria turn blue &

nonrecombinant are white

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SelectionSelection Selection is a screening

process designed to facilitate the

identification of recombinantbacteria while preventing growth

of nontransformed bacteria (or

those containing plasmid without

foreign DNA) Blue-white screening is

becoming more popular (uses ß-

galactosidase)

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Antibiotic selection Antibiotic selection

Antibiotic selection uses a

plasmid vector with genes

encoding resistance to 2different antibiotics, usually

ampicillin (ampR) and

tetracycline (tetR)

Foreign DNA inserted into one of the 2 antibiotic

resistance genes (disrupts gene - preventing protein)

Transformed cells are plated to an agar plate with no

antibiotic or plate with one (ampicillin)

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Replica platingReplica plating Replica plating uses

sterile pads pressed against

colonies on plate (cells

adhere to make an exact

copy)

Then pad is placed on 2nd

replica plate containing 2ndantibiotic (tetracycline)

Nontransformed bacteria cannot grow in presence of either

antibiotic without plasmid

Compare plates since recombinant can¶t grow on 2nd plate

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Replica plating diagramReplica plating diagram

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Vector FeaturesVector Features

5. RNA polymerase promoter

sequences - place where RNA

polymerase binds to begintranscription

6. DNA sequencing primer

sequences - known sequence

that allows sequencing of

cloned DNA fragments that have

been inserted into the plasmid

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Types of VectorsTypes of VectorsOne primary limit ation 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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Gene Transfer Gene Transfer Cohen discovered that plasmid

DNA enters a bacterial cell

(transformation) treated with

c alcium chloride, chilled on ice,then briefly heated

A more recent transformation

technique is electroporation (brief

pulse of high-voltage electricity tocreate tiny holes in bacteri al cell

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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BiolisticsBiolistics 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 gold particles with DNA attached

Done with a gene gun (akabioblaster )

Can be used on bacteria, yeasts,

& mammalian cell lines

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National Institutes of Health

(NIH)

National Institutes of Health

(NIH)

Concerns arose

because of new

techniques

In 1975, NIH formed

the Recombinant DNA

AdvisoryC

ommittee(RAC) to evaluate risks

and establish guidelines

for rDNA technology

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