LBA 11)

Polumerase Chain reaction

• Make copies of DNA in your sample

GMO crops

• NOT Selective breading

• MUSTARD SEEDS

  • Kale, brussel sprots, broccoli, cabbage, and cauliflower

HT Gene

• Herbicide tolerant to glyphasat chemical

• Bt corn and cotton

Glyphasat

• A common component found in powerful herbicides (weed killers)

Bt endotoxin gene delta

• BacilliusThuringiensis

• A spore forming bacteria that produces insecticidal proteins that kill some insects

• Harmless to humans

Opposition to Bt endotoxin gene

• Harmful to beneficial insect

• Produce super bugs

• 26 countries bann

Creating GMO plants

• Gene must go into selected chromosme

1. Isolation of Ti-plasmid from A.tumerfaciens

2. Replace T-DNA with gene of interest

3. Transformation of A.Tumefaciesn with modified Ti plamsi w/gen of interest

4. Infect plant with transformed A.tumefacien

A. tumefaciens

• Tumor inducing bacterium

• Ti-plasmid in the bacterium has T-DNA (transfer DNA)

• T-DNA from Ti plasmid is transferred into the DNA of the host plant cell

T-DNA

• Tranfer DNA

• Indid eof Ti plasmid from A.Tumegaciens

Polymerase Chaim reaction

• Isolate specific DNA fragment and make many copies of the gene in test tubes

• DNA strands separe using HEAT to break H-bonds

• NOOO primase

  • Primers are made of DNA NOTTTT RNA

  • 2 primers 1 for each strand

Primers in PCR

• They are made of DNA

• There is one for each strand

Helicase

• Separate the 2 h-bonded strand of DNA

DNA polumerase

• Extends teh RNA primer created by primase at the 3’ end

Steps to doing PCR LONG

1. Many copies of the 2 DNA primers

2. Many copied of target DNA

3. A mixture of 3 DNA nucleotides

   1. dATP, dCTP. dGTP, dTTP

4. Add Taq DNA polymerase

5. Heat to 95C to denature

6. Cool at 60 C to anneal primers bond to their target complementary DNA sequences

7. Taq DNA polymerase extends primers in 5’ → 3’ direction

Heating and Coolin

• Heating 30 sec at 95C

  • Denatures DNA

• Annealing 50-60C for 1-2 mins

  • Too high = primers wont anneal to target DNA

  • Too low = primers anneal to incorrect region

• Replication 72 C 1 -3 mins

  • Optimal for Taq DNA polymerase

Taq DNA polumerse

• Heat resistanct DNA polymerase

Cycle Time

• Repeats 30 -40 times in a sing PCR (1.5 hours)

• Target sequence amplified exponential

• Non target sequence amplified linearly

Instagene Matrix

• Negatively charged microscopic beads that bind cation like Mg2+

• The boilding process releases DNases and Mg2+ ions

• Negatively charged microscopic beads bind to Mg2+ ions which are required as co-factors to activate DNase

Lab

• Mortar and Pestle w/water

• Centrufugate at 95C

  • Pelate = non-DNA cellular material

  • Supernantant = DNA remaining

• Add 2 primers to the isolated DNA samples

  • P-primers

  • G-primers

P Primers

• 450 base pair region of gene for photosysthesis

G-Primers

• 200 bp region of Bt delta endotoxin gene