1. Manipulating DNA: tools and techniques
Chapter 12
Key Knowledge:
tools and techniques: gel electrophoresis; DNA profiling; DNA sequencing; DNA recombination; DNA amplification; gene cloning, gene transformation; gene delivery systems;

2. Mitochondrial DNA (mtDNA)
Inherited maternally
Behaves like prokaryotic DNA
Haploid
Circular
More than one copy in the mitochondria
Useful in family studies
mtDNA D-Loop extremely variable, accumulates many different mutations because it is not transcribed

4. Mitochondrial DNA (mtDNA)
see Pages 419 – 421 of textbook “Murders at Ekaterinberg”.

5. Tools for Genetic Engineering
Action
‘Tool’
Cut DNA into fragments at precise locations
Restriction Enzymes
(pp. 422 – 423)
Separate fragments by size
Electrophoresis
(pp. 423 – 424)
Find particular DNA fragments
Probes
(pp. 424 – 425)
Join DNA fragments
Ligase Enzyme
(pp. 425 – 426)
Transport DNA into cells
Vectors (p. 426)
Obtain multiple copies of gene
Gene Cloning
(p. 428)

6. Restriction Enzymes
The molecular biology revolution started with the discovery of restriction enzymes (restriction endonucleases)
Enzymes cleave DNA at specific sites
These enzymes are significant in two ways
Allow a form of physical mapping that was previously impossible
Allow the creation of recombinant DNA molecules (from two different sources)

7. Restriction Enzymes Restriction enzymes cut at specific sites
Some form ‘blunt ends’ (straight cut) others form ‘sticky ends’ (staggered cut). e.g. EcoRI

9. Gel Electrophoresis A technique used to separate DNA fragments by size
The gel (agarose or polyacrylamide) is subjected to an electrical field
The DNA, which is negatively-charged, migrates towards the positive pole
The larger the DNA fragment, the slower it will move through the gel matrix
DNA is visualised using fluorescent dyes.

10. Dye added to the DNA
Makes the sample visible when it is put into the agarose wells

11. Buffer solution added to the tank
This ensures that the electric current goes through the whole tank and that maintains that ions can move in the solution

12. DNA samples loaded into wells
Glycerol also in the loading dye

13. Electrical current applied to the chamber
Safety cover is put over the top and the current is switched on
The dye will migrate through the gel toward the positive electrode, as will the DNA
Depending on how much voltage is applied and how warm the gel is and size and shape of molecules will depend on how fast the mols move through the gel
Smaller fragments will move easier so they will be closer to the positive electrode
Once the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the tray

14. DNA is stained using ethidium bromide
Gel is stained using ethidium bromide which binds to DNA it shows up as bands in UV light
Draw attention to the fact that small mols are at the bottom of the gel and large ones stay nearest to the wells

15. Probes
A probe is used in order to find a specific sequence of DNA within a relatively large sample.
Probes are usually labelled with a marker (e.g. radioactive, fluorescent, etc.) and are complementary to the target sequence.
See Figure 12.9 in your textbook (pg 424).

17. Ligase
The enzyme Ligase is required to catalyse the joining of pieces of dsDNA at their sugar-phosphate backbone.

19. Transporting DNA into cells
DNA can be transported into cells through the use of vectors.
Vectors are cellular agents that have the capacity to carry DNA and transport it into target cells.
Bacteria are commonly used as vectors.
Bacteria have a small circular piece of DNA called a plasmid.

20. Steps to Transporting DNA into a Target Cell
DNA of the plasmid is cut using a restriction enzyme (the same restriction enzyme that the original DNA is cut with).
The plasmid and the foreign DNA are mixed and their ‘sticky ends’ pair
DNA ligase makes the joins permanent.
The plasmids that contain the recombinant DNA plasmid are then selected.

21. Steps to Transporting DNA into a Target Cell

22. Gene Isolation
It is easy to isolate the total DNA from human somatic cells
46 chromosomes
6 x 109 base pairs of DNA
20,00 – 25,000 genes
Sequences of non-coding DNA

23. Gene Isolation
Harder to isolate one gene or part of a gene

24. Gene Isolation
Locate particular DNA fragments following separation by electrophoresis using a probe with a complementary base sequence

25. Gene Isolation
Synthesis DNA from nucleotides: Use a DNA synthesiser to artificially manufacture a specific DNA sequence with lengths greater than 50 bases to be used as primers or probes

27. Gene Isolation
Make a copy of DNA using a mRNA template: mRNA is isolated from specific cells and the enzyme Reverse Transcriptase is used to a make a complementary ssDNA strand. This is called copy DNA (cDNA).
DNA Polymerase may be used later to convert the cDNA into dsDNA.
This method only works for coding DNA: genes that produce mRNA

29. Gene Cloning Making multiple copies of a gene
The gene is copied and placed into a bacterial plasmid
The plasmid is inserted into the bacterial host cell
Inside bacterial host cell the plasmid and the gene make twenty copies of itself
The bacterial cell copies itself every twenty minutes by binary fission
Within several hours there are millions of copies of the bacterial host cell and the inserted gene.
In some case the gene is switched on and the product (protein) is harvested.

30. Polymerase Chain Reaction (PCR)
Polymerase chain reaction enables large amounts of DNA to be produced from very small samples.
There is a repeating cycle of:
Separation of double DNA strands
Annealing of primers to the sequence to be amplified.
Extension of DNA using the original DNA strand as a template.

31. The Reaction
PCR tube
THERMOCYCLER

32. Separation (heat to 95oC)
Lower temperature to 56oC - Anneal with primers
Increase temperature to 72oC - DNA polymerase + dNTPs = extension of DNA

34. Gene Transformation First identified by Griffith in 1928
Defined as the take up of naked DNA by cells
Occurs naturally in bacteria, yeast and some plants
May be induced
Mix bacteria with CaCl2 solution
Place in ice (0°C)
Then place at 37°C
Cells are now competent

37. Gene Delivery Systems
Gene Delivery is a process of inserting foreign DNA into host cells
There are many different processes
Viral
Non-viral
It is the key to Gene Therapy

38. Gene Delivery Systems – Viral
Viral vectors
They are good at targeting and entering cells
Some can be engineered to target specific types of cells
They can be modified, so they do not replicate and destroy the cell

39. Gene Delivery Systems – Non Viral
Plasmids
microinjection
gene gun
impalefection
hydrostatic pressure
electroporation
continuous infusion
sonication
lipofection