separately - Rossmant

AP Genetic Modification Continued

•
Objectives:

–
Determine how organisms actually become
transgenic (genetically modified).

–
See how gel electrophoresis allows you to
determine the genetic fingerprint.

•
Bell Work: Quick Poll

–
Do you have
netfix
?

•
Homework: Watch Food Inc on Netflix

http://www.youtube.com/watch?v=PSwlCk_Z0
2c

gel electrophoresis animation

http://www.youtube.com/watch?v=yta5KC18
WkU&NR=1&feature=endscreen

how to clone
–

review


http://www.youtube.com/watch?v=ZxWXCT9
wVoI

Closure
–

gel
electophoresis

uses

http://learn.genetics.utah.edu/content/labs/g
el/

virtual lab

Chimeras

•
Cohen and Boyer’s Chimera

•
Take bacterial plasmid

•
Cut with
EcoRI

•
Take 9000
bp

fragment

•
Combine the ends of the fragment into a smaller
plasmid = pSC101

Replication start point

Resistance gene for
antibiotic tetracycline

Chimera = New genome that would
never have existed without humans =
Recombinant DNA

pSC101

Chimeras

•
Cohen and Boyer’s Chimera

•
Take bacterial plasmid

•
Cut with
EcoRI

•
Take 9000
bp

fragment

•
Combine the ends of the fragment into a smaller
plasmid = pSC101

Replication start point

Resistance gene for
antibiotic tetracycline

Chimera = New genome that would
never have existed without humans =
Recombinant DAN

How does it
combine into a
smaller circle
after being cut
with
EcoRI
?

Manipulate pSC101

•
Use
EcoRI

to cut DNA from frog that coded for
rRNA

–
Open pSC101

–
Add frog
rRNA

gene

–
Add bacteria

•
Now, the bacteria that takes up the plasmid
that resisted tetracycline is also making frog
rRNA
.

Vectors

•
Plasmids can be induced to make hundreds of
copies with their foreign genes.

•
Can even use artificial chromosomes as
vectors.

•
Or use a virus.

Vector = The genome that
carries the foreign DNA into a
host.

HOST CELL

Why do
viruses
make good
vectors?

Real Life GM

•
Bulls with gene for human antibacterial and
iron transport.

Herman

Some of his calves carry the
gene too.


Transgenic herd capability?


Real Life GM

•
Wilt Proof Flowers

–
Ethylene makes flowers wilt

–
Make flower insensitive to ethylene = no wilting!

Real Life GM

•
Transgenic Salmon

Salmon

Embryo

Growth
Hormone

Shortens reproductive
cycle

Makes salmon 11x
bigger!

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of recombinant DNA

3.
Cloning

4.
Screening

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of
recombinant
DNA

3. Cloning

4. Screening

a)
Cut with a restriction
endonuclease

into
fragments.

i.
Different
endonuclease

=
different fragments

ii.
Can separate using gel
electrophoresis.

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3. Cloning

4. Screening

a)
Cut with a restriction
endonuclease

into fragments.

i.
Different
endonuclease

=
different fragments

ii.
Can separate using gel
electrophoresis.
-

demonstration

Gel Electrophoresis
–

a
process of separating DNA by
size.

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3. Cloning

4.

Screening

Cut with a restriction
endonuclease

into
fragments.

a)
Different
endonuclease

=
different fragments

b)
Can separate using gel
electrophoresis.

Fragments of DNA are inserted into
plasmids or viral vectors.

Why is it
important to use
the same
restriction
endonuclease
?

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3.

Cloning

4.

Screening

Cut with a restriction
endonuclease

into
fragments.

a)
Different
endonuclease

=
different fragments

b)
Can separate using gel
electrophoresis.

Fragments of DNA are inserted into
plasmids or viral vectors.

Vector is are inserted into cells
(usually bacteria).

a)
These are maintained
separately

in clone libraries.

b)
Some may have taken the
wrong vector or not have
taken one at all.

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3.

Cloning

4.

Screening

Cut with a restriction
endonuclease

into
fragments.

a)
Different
endonuclease

=
different fragments

b)
Can separate using gel
electrophoresis.

Fragments of DNA are inserted into
plasmids or viral vectors.

Vector is are inserted into cells
(usually bacteria).

a)
These are maintained
separately

in clone libraries.

b)
Some may have taken the
wrong vector or not have
taken one at all.

Screen the library to find the
fragment of interest.

VERY challenging.

Have to get rid of clones without
vectors and clones that are lacking
the wanted vectors.


Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3.

Cloning

4.

Screening

Screen the library to find the
fragment of interest.

VERY challenging.

Have to get rid of clones without
vectors and clones that are lacking
the wanted vectors.

Eliminate cells without vectors by
placing in an antibiotic.


Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3.

Cloning

4.

Screening

Screen the library to find the
fragment of interest.

VERY challenging.

Have to get rid of clones without
vectors and clones that are lacking
the wanted vectors.

Eliminate cells without vectors by
placing in an antibiotic.


Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3.

Cloning

4.

Screening

Screen the library to find the
fragment of interest.

VERY challenging.

Have to get rid of clones without
vectors and clones that are lacking
the wanted vectors.

Eliminate cells without vectors by
placing in an antibiotic.


We can also use the
Lac Z
gene


Lac Z

•
Helps cells metabolize specific sugar (x gal)

•
When Lac Z metabolizes x gal it creates a blue product.

•
SO… We can use a restriction enzyme that cuts Lac Z. So
the bacteria will live in antibiotics and NOT produce blue
product.

Bacterial cell that did
not take up plasmid

Functional
lac

z gene

Lac Z gene Non Functional

Fragment of DNA

Lac Z gene Functional

No wanted fragment of
DNA uptake

Antibiotic
Resistance
gene

Place in antibiotic. Plasmids without
antibiotic resistance will die.

This tells us if a plasmid was picked up.

It still doesn’t tell us if the plasmid has the
DNA we want.

Put in X
-
gal sugar solution. The bacteria
that have the plasmid with the correct DNA
will NOT produce blue since the
LacZ

has
been cut.

Steps to Genetic Engineering

1.
DNA cleavage

2.
Production of



recombinant DNA

3.

Cloning

4.

Screening

Screen the library to find the
fragment of interest.

VERY challenging.

Have to get rid of clones without
vectors and clones that are lacking
the wanted vectors.

Eliminate cells without vectors by
placing in an antibiotic.

Then in
Xgal

to see if they turn blue or
not.


Clone Libraries

•
They are HUGE!

•
Many fragments of DNA possible.

•
If you want to find a particular sequence on a
particular fragment you must hybridize.

Hybridization

•
Take colonies from clone libraries.

Hybridization

•
Take colonies from clone libraries.

•
Make a replica with a filter.

Hybridization

•
Take colonies from clone libraries.

•
Make a replica with a filter.

Hybridization

•
Take colonies from clone libraries.

•
Make a replica with a filter.

•
Wash to denature the DNA and include
radioactive labeled probes.

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

Hybridization

•
Only the colonies with that gene
will retain the probe and emit
radioactivity on a film placed over
the filter = autoradiography

Hybridization

•
Compare to the original plate
to find the colony of interest.

How do we get lots of copies of that
gene that we’ve found and we want?

•
Old fashioned = bacterial implant = slow =
unreliable.

•
NEW AND WONDERFUL




Polymerase Chain Reactions!!!