Biotechnology

2015

BIG IDEA

• Technology can be used to alter DNA and test DNA.

Restriction Enzymes

• Can’t do much with DNA in Biotech unless we can CUT DNA up… Restriction Enzymes do the job!

• Watch video from start-7:00 mins

Restriction enzymes• Cut DNA at specific sites– Look for palendrome sequence (restriction site)– leave “sticky ends”

GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA

GTAAACGAATTCACGCTTCATTTGCTTAAGTGCGAA

restriction enzyme cut site

restriction enzyme cut site

Named

Many different enzymes◦named after organism they are

found in (ex: restriction enzyme 1 in e. coli bacteria…EcoRI)

EcoRI, HindIII, BamHI, SmaI

IMPT: TAQ polymerase

Many uses of restriction enzymes…

• Now that we can cut DNA with restriction enzymes…– we can cut up DNA from different people… or

different organisms… and compare it or splice new combinations together

– why?• forensics• medical diagnostics• paternity• evolutionary relationships • Destroying cancer cells• Making medications• and more…

Restriction enzymes + Bacteria

• Bacterial Review

Restriction Enzymes + Bacteria

• Bacteria review – one-celled prokaryotes– reproduce by binary fission– rapid growth• generation every ~20 minutes• 108 (100 million) colony overnight!

– incredibly diverse

Plasmids • In addition to 1 circular chromosome, bacteria also have

small supplemental circles of DNA called plasmids– FXN: carry extra genes

• 2-30 genes • genes for antibiotic resistance• self-replicating

– Plasmids can be exchanged between bacteria• “bacterial sex”

– can be imported from environment (transformation…remember Griffiths Experiment?)

plasmids

• Used as vectors to insert new genes into bacteria

plasmid

cut DNA

gene fromother organism

glue DNA

recombinantplasmid

vector

transformedbacteria

Bacteria express the new gene

Medical Implications

• What would happen if we could splice in the gene for insulin production into bacteria…

• Video

• Only possible because of restriction enzymes

PCR• Kary Mullins: development of PCR technique– a copying machine for DNA

1985

PCR

• Polymerase Chain Reaction– method for making

many, many copies of a specific segment of DNA

– only need 1 cell with DNA to start

– WHY? Sometimes the DNA sample you want to run tests on is too small (crime scene)

pcr

• It’s copying DNA in a test tube!

• What do you need?– template strand– DNA polymerase enzyme– Nucleotides (triphosphate

form)• ATP, GTP, CTP, TTP

– primer

http://www.sumanasinc.com/webcontent/animations/content/pcr.html Thermocycler

PCR process

• Cycle 1– Heat/Denature H bonds– Cool (Anneling)– Extension– End: 2 DNA

• Cycle 2– Same as 1– End: 4 DNA

• Cycle 3– Same as 1 – End: 8 DNA

PCR20-30 cycles3 steps/cycle30 sec/step

The polymerase problem• In step 1 must heat DNA to denature– 90°C destroys DNA polymerase so can’t use this to

bind in new nucleotides like normal– have to add new enzyme every cycle• almost impractical!

• Need enzyme that can withstand 90°C…– Taq polymerase• Enzyme found in bacteria living in hot springs– Thermus aquaticus

DNA FINGERPRINTING

STR’S AND FINGERPRINTS

• BEFORE starting the notes on this content, watch this video:

http://www.youtube.com/watch?v=DbR9xMXuK7c

Comparing cut up DNA• How do we compare DNA fragments?– separate fragments by size

• How do we separate DNA fragments?– run it through a gelatin• agarose• made from algae

– Process called: gel electrophoresis

Gel Electrophoresis• A method of separating

DNA in a gelatin-like material using an electrical field– Phosphate end of each

NT is negatively charged

– DNA moves toward the positive side

– Smaller fragments travel further

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

Uses: Medical diagnostic

• Comparing normal allele to disease allele

chromosome with disease-causing

allele 2

chromosomewith normal

allele 1 –

+

allele 1allele 2

DNA

Example: test for Huntington’s disease

Uses: Forensics• Comparing DNA sample from crime scene

with suspects & victim

–

+

S1

DNA MOVES

S2 S3 Vsuspects crime

scene sample

Uses: Paternity

• Who’s yo daddy?

+

DNA

childMom F1 F2–

Uses of genetic engineering

• Genetically modified organisms (GMO)– enabling plants to produce new proteins• Protect crops from insects: BT corn

– corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)

• Extend growing season: fishberries – strawberries with an anti-freezing gene from flounder

• Improve quality of food: golden rice – rice producing vitamin A

improves nutritional value

GMO research

• Follow directions on notes sheet…

CLONING

• Follow directions on notes sheet (in class if time OR homework)

• Link 1: http://learn.genetics.utah.edu/content/tech/cloning/whatiscloning/

• Link 2: http://learn.genetics.utah.edu/content/tech/cloning/clickandclone/