1. BIOTECHNOLOGY

2. What is biotechnology? Aspect of technology that uses: - biological data - molecules - organisms for alternative practices

3. Bioremediation The use of living microorganisms to transform harmful substance into non-toxic compounds Example: EXXON VALDEZ oil spill in March 1989 – 50,000,000L of oil spewed into the Alaskan Sea – Covering 3,400km 2

5. Exxon Valdez Clean-up Used: – Physical barriers – Pumps – *microrobes Scientists released microbes with oil degrading enzymes – Research is now looking into adding nutrients (O2) to the oil spill to increase microbe growth

6. Uses of Biotechnology 1.Investigating genetic disorders 2.Altering genetic make-up of organisms - production of useful proteins 3. Analyze DNA evidence

7. Biotechnology Tools Like with any trade, there are certain tools needed to complete a specific task Biotechnologists, or molecular biologists, use biological molecules – to cut, join, & replicate DNA

8. Biotechnology Tools 1.Restriction Endonucleases / enzymes 2.Methylases 3.DNA ligase 4.Gel Electrophoresis 5.Plasmids 6.Transformation 7.PCR 8.RFLPs 9.DNA sequencing

9. 1. Restriction Endonucleases (RE’s) AKA Restriction Enzymes (RE’s) Enzymes that are able to cleave (cut) double stranded DNA into fragments – Only cut at specific base pairs Each RE has its own recognition site – Specific DNA sequence – 4-8 base pairs in length – Characterized by a palindromic sequence

10. 1. Restriction Endonucleases (RE’s) Ex. EcoRI is a restriction enzyme Cuts DNA when it sees the following sequence 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ **Considered palindromic because both strands have the same sequence when read 5’  3’

11. 1. Restriction Endonucleases (RE’s) How RE’s work (text form) 1.Scan DNA for cognition site 2.Once found, it binds and uses a hydrolysis rxn to break phosphodiester linkages between A and G nucleotides on each strand 3.H-bonding between nucleotides is distrupted 4.Two fragments are produced

12. 1. Restriction Endonucleases (RE’s) The ‘ends’ produced by RE’s depend on what RE is used. There are two types of ‘ends’: 1.Sticky Ends 2.Blunt Ends

13. 1. Restriction Endonucleases (RE’s) Sticky Ends fragment end has short SS DNA with unbound base pairs Generally more useful – Easier to join to other sticky ends Ex. EcoRI 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’ 5’ – G A A T T C – 3’ 3’ – C T T A A G – 5’

14. 1. Restriction Endonucleases (RE’s) Blunt Ends Fragment ends are fully base paired Less useful – as harder to bind to other blunt ends Ex. SmaI 5’ – G G G C C C – 3’ 3’ – C C C G G G – 5’ 5’ – G G G C C C – 3’ 3’ – C C C G G G – 5’

15. 1. Restriction Endonucleases (RE’s) Question: Why would it be better to have 4-8 base pairs in your recognition site than 2 base pairs? Answer: The shorter the recognition sequence, the more likely the RE is to cut & may disrupt a gene *probability of finding a 6bp recognition site: 4 x 4 x 4 x 4 x 4 x 4 = 4,096 1 in 4,096 bases.