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 minsvideo

Restriction enzymes Cut DNA at specific sites – Look for palendrome sequence (restriction site) – leave “sticky ends” GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA GTAAACGAATTCACGCTT CATTTGCTTAAGTGCGAA 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

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 10 8 (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 from other organism glue DNA recombinant plasmid vector transformed bacteria 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

Kary Mullins: development of PCR technique – a copying machine for DNA 1985

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 Thermocycler

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 PCR cycles 3 steps/cycle 30 sec/step

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: 7c

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 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

Comparing normal allele to disease allele chromosome with disease-causing allele 2 chromosome with normal allele 1 – + allele 1 allele 2 DNA  Example: test for Huntington’s disease

Comparing DNA sample from crime scene with suspects & victim – + S1 DNA MOVES  S2S3V suspects crime scene sample

Who’s yo daddy? + DNA  childMomF1F2 –

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: loning/whatiscloning/ loning/whatiscloning/ Link 2: loning/clickandclone/ loning/clickandclone/