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_Z02c gel electrophoresis animation

http://www.youtube.com/watch?v=yta5KC18WkU&NR=1&feature=endscreen how to clone – review

http://www.youtube.com/watch?v=ZxWXCT9wVoI Closure – gel electophoresis uses

http://learn.genetics.utah.edu/content/labs/gel/ 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

SalmonEmbryo

Growth Hormone

Shortens reproductive cycle

Makes salmon 11x bigger!

Steps to Genetic Engineering

1. DNA cleavage2. Production of recombinant DNA

3. Cloning4. Screening

Steps to Genetic Engineering1. DNA cleavage2. Production of

recombinant DNA

3. Cloning4. Screening

a)Cut with a restriction endonuclease into fragments.i. Different endonuclease =

different fragmentsii. Can separate using gel

electrophoresis.

Steps to Genetic Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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

Func

tiona

l la

c z

gene

Lac Z gene Non FunctionalFragment 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 Engineering1. DNA cleavage2. Production of recombinant DNA3. Cloning4. 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!!!

PCR