BIOTECHNOLOGY

What is biotechnology?

• Aspect of technology that uses:- biological data- molecules - organisms for alternative practices

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 3,400km2

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

Uses of Biotechnology1. Investigating genetic disorders

2. Altering genetic make-up of organisms- production of useful proteins

3. Analyze DNA evidence

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

Biotechnology Tools1. Restriction Endonucleases / enzymes2. Methylases3. DNA ligase4. Gel Electrophoresis5. Plasmids6. Transformation7. PCR8. RFLPs9. DNA sequencing

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

1. Restriction Endonucleases (RE’s)

Ex. EcoRI is a restriction enzymeCuts 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’

1. Restriction Endonucleases (RE’s)

How RE’s work (text form)1.Scan DNA for cognition site2.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 distrupted4.Two fragments are produced

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 Ends2.Blunt Ends

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’

1. Restriction Endonucleases (RE’s)

Blunt Ends• Fragment ends are fully base paired• Less useful

– as harder to bind to other blunt ends

• Ex. SmaI5’ – 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’

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.