1. The use of living things, biological systems and processes for the benefit of humans.

2. Tools for manipulating DNA
1. CUTTING
DNA into fragments using restriction enzymes: these molecules cut at specific DNA sequences and are only found in prokaryotic organisms
2. PASTING
Pasting DNA fragments together using enzymes called ligases. We can join fragments of DNA to make what is called “recombinant DNA”. DNA ligases found in many species including humans.
3. COPYING
Making many copies of DNA (amplification) using DNA polymerases in the Polymerase Chain Reaction (PCR) technique. All organisms contain DNA polymerases as they all need to copy their DNA.
4. TRANSFERRING
DNA into cells using vectors such as “plasmids”. This technique is called a transformation in prokaryotic cells.

3. 1. Cutting DNA
Restriction enzymes (molecular scissors) are found in prokaryotic organisms
Cut DNA into smaller pieces (restriction fragments) in a controlled way – specific sequences of DNA called recognition sites.

4. Specificity Restriction enzymes are specific:
The DNA and the enzyme need to be mixed together and incubated at a temperature that will result in maximum activity of the enzyme.
Each restriction enzyme will only cut the DNA at a specific sequence of A, G, T and Cs. We call this place a recognition site.

5. Different restriction enzymes recognise specific recognition sites.

6. Cutting Specificity GAATTC CTTAAG GTT AAC GTTAAC CAA TTG CAA TTG
When DNA is cut with a restriction enzyme the resulting fragments are left with either a short overhang of single stranded DNA called a sticky end or no overhanging DNA which is called a blunt end (snake demo)
EcoRI – leaves sticky ends
GAATTC
CTTAAG
HpaI – leaves blunt ends
GTT AAC
GTTAAC
CAA TTG
CAA TTG
- site where enzymes cuts through the sugar phosphate backbone of the DNA strand.

7. What’s in a name!
Restriction enzymes are named after the organism from which they were isolated.
E.g. Escherichia coli
EcoRI
The Roman number indicates the order of discovery
If another letter is placed in front of the Roman number it signifies a particular strain of the bacterium. R = resistance

8. Restriction Enzyme
Restriction Site
Overhang Type
EcoRI
E = genus Escherichia
co = species coli
R = strain RY13
I = first endonuclease isolated
GAATTC
CTTAAG
STICKY
BamHI
B = genus Bacillus
am = species amyloliquefaciens
H = strain H
GGATCC
CCTAGG
HindIII
H = genus Haemophilus
in = species influenzae
d = strain Rd
I = third endonuclease isolated
AAGCTT
TTCGAA
HpaI
pa = species parainfluenzae
GTTAAC
CAATTG
BLUNT

9. Fragments are sorted by Gel Electrophoresis
This technique is used to separate out fragments, obtained by a restriction digest, of DNA according to their size (length in base pairs).
DNA fragments are separated into bands containing fragments of the same length by electrical separation in a gel matrix.
DNA molecules migrate to the positive electrode, when an electric field is applied to the gel matrix, as they are negatively charged.
This technique is used to isolate DNA fragments containing genes which are subsequently used to make recombinant DNA.

10. Fragments are sorted by Gel Electrophoresis

11. 2. Pasting When two samples of DNA are combined using DNA ligases.
Any 2 DNA strands can be joined that have complementary exposed nucleotides (i.e. cut with same restriction enzyme).

12. 3. Copying (Amplification) of DNA using the Polymerase Chain Reaction
The $300 million dollar man.
“I was working for Cetus, making oligonucleotides (primers). They were heady times. Biotechnology was in flower and one spring night while the California buckeyes were also in flower I came across the polymerase chain reaction. It was the first day of the rest of my life”. Kary Mullis 1972

13. Why PCR? To amplify a small amount of DNA into an analysable quantity
E.g. crime scene, fossils etc

14. PCR Tools
Taq DNA Polymerase – is an enzyme that works well at 72°C.

15. PCR Tools
Primers:
Synthetic short segments of DNA up to 25 nucleotides long.
Probe for a specific sequence or gene along a strand of DNA.
Hybridise with a sequence of bases on the template DNA through complementary base pairing.
Indicate to Taq DNA polymerase where to start building the complementary strand by extending the primer.

16. Find the starting point for copying STR regions
Select your primer Start region Sequence to be copied by extending the primer.

17. Thermocycling machine
Step 1. Denaturation
Step 1: Denaturing the DNA – 2 minutes
T A C C G T A A
A T G C C A T T
Thermocycling machine
Step 1: 92°C
At this temperature the hydrogen bonds are broken resulting in two single strands of DNA.

18. Step 2. Attachment of Primers
T A C C G T A A
A T G
T A A
A T G C C A T T
Step 2: 55°C
Step 2: Primer annealing– 2 minutes
The temperature is lowered to allow the primers to bind (anneal) to their complementary bases on each of the single strands of DNA.

19. Step 3: Extension Step 3: DNA synthesis – 1 minute
T A C C G T A A
A T G G C
Taq A T T A T C
Step 3: 72°C
Taq
G G T A A
A T G C C A T T
Step 3: DNA synthesis – 1 minute
Taq DNA polymerase extends the DNA strand from the primers using the base pairing rule.

20. And you can repeat the three step cycle over and over!

21. PCR song

22. Fluorescent jellyfish
4. Transferring
Jellyfish
Fluorescent
Because DNA is the same in all organisms, we should be able to take a piece of DNA from one organism and put it into another organism.
You can change the way an organism looks or behaves!
This process of taking DNA from one organism and putting into another is called transformation.
Plasmid
Jellyfish and plasmid DNA is cut with the same restriction enzyme.

23. Vectors
Gene inserted into a vector that will carry the gene into the desired organism.
Common vectors are:
Viral vectors (eg. Adenovirus and retorovirus) – must have disease symptom genes removed first!
Liposome vectors – small circular molecules surrounded by phospholipid bilayer
Plasmid vectors – small circular piece of bacterial DNA. Plasmids are used as vectors in bacterial transformations.

24. Plasmids are not naturally attracted to bacteria!
Bacterium

25. Transformation of Bacteria with a Recombinant DNA Plasmid
Making the bacteria more ‘attractive’ to plasmids Plasmids are now attracted to the bacteria
Bacterium
CaCl2 solution

26. The Transformation
Now give the bacteria some food and the right temperature to reproduce.
Any bacteria with the plasmid inside will start making the jelly protein, that results in fluorescence.
HEAT SHOCK

27. Gene Sequencing
Gene sequencing is identifying the nucleotide order in a segment of RNA or DNA.
A G G A C T C A T G G A G A A G A A C T T T . . .
Our genome has been sequenced. We have 3,100,000,000 base pairs, what a big book!

28. Gene Cloning
Making identical copies of sequences of DNA that code for proteins using plasmids
extract plasmid from bacteria
Cut plasmid DNA and DNA of the gene to be inserted with same restriction enzyme
Paste 2 pieces of DNA using DNA ligase to create a recombinant plasmid.
Add recombinant plasmid to bacterial culture, where some are taken up and replicate (called transformation)
Isolate and analyse bacteria containing recombinant plasmids.
PRACTICAL APPLICATION: Production of human growth hormone

29. DNA Profiling Compares base sequence of 2 or more individuals
Short tandem repeats (STRs) and variable nucleotide tandem repeats (VNTRs): non-coding sections of DNA repeated many times between genes
E.g. GAGAGAGAGAGAGA
There are more than 10,000 STR loci in one set of human chromosomes!

30. DNA Profiling
The repeat is present in all members of the population, but the number of repeats varies among individuals and is inherited.
DNA profiling allows us to view these patterns in our DNA.
Uses PCR and gel electrophoresis – smaller fragments will migrate further on the gel.

31. DNA Profiling

32. DNA Profiling
Loci of STR regions found to vary from person to person with a high frequency
13 are used in America, but only 9 are used in Australia – why?